Dermal fillers comprising silk fibroin hydrogels and uses thereof

ABSTRACT

The present specification provides compositions useful as dermal fillers and methods using such compositions to treat a condition of skin.

CROSS REFERENCE

This patent application is a continuation-in-part that claims priorityunder 35 U.S.C. §120 to U.S. Non-Provisional patent application Ser. No.12/764,039, filed Apr. 20, 2010, a patent application that claimspriority pursuant to 35 U.S.C. §119(e) to U. S. Provisional PatentApplication Ser. No. 61/170,895 filed Apr. 20, 2009, each of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present specification discloses purified silk fibroin and method forpurifying silk fibroins, hydrogels comprising silk fibroin with orwithout an amphiphilic peptide and methods for making hydrogelscomprising silk fibroin and the use of silk fibroin hydrogels in avariety of medical uses, including, without limitation fillers fortissue space, templates for tissue reconstruction or regeneration,scaffolds for cells in tissue engineering applications and for diseasemodels, a surface coating to improve medical device function, or as aplatform for drug delivery.

BACKGROUND

Silk refers to a filamentous product secreted by an organism such as aspider or silkworm. Fibroin is the primary structural component of silk.It is composed of monomeric units comprising an about 350 kDa heavychain and an about 25 kDa light chain, and interspersed within thefibroin monomers is another about 25 kDa protein derived from the P25gene. The ratio of heavy chain:light chain:P25 protein is about 6:6:1.Fibroin is secreted by the silk glands of the organism as a pair ofcomplementary fibrils called “brins”. As fibroin brins leave the glands,they are coated with sericin, a glue-like substance which binds thebrins together. Sericin is often antigenic and may be associated with anadverse tissue reaction when sericin-containing silk is implanted invivo.

Silkworm silk fibers traditionally available in the commercial marketare often termed “degummed”, which refers to the loosening and removalof a portion of the sericin coat surrounding the two fibroin brinsthrough washing or extraction in hot soapy water. This degummed silkoften contains or is recoated with sericin and other impurities in orderto bind the plied multifilament together into a single fiber. Therefore,degummed silk, unless explicitly stated to the contrary, typicallycontains twenty percent to twenty-eight percent (by weight) sericin andcan not be assumed to be sericin-free.

Silk fibers have historically been valued in surgery for theirmechanical properties, particularly in the form of braided filamentsused as a suture material. Residual sericin that may be contained inthese materials stands as a potential obstacle to its use as abiomaterial as it does present the possibility for a heightened immuneresponse. This sericin contamination may be substantially removedthough, resulting in a virtually sericin-free fibroin which may be usedeither as fibers or dissolved and reconstituted in a number of forms.For example, natural silk from the silkworm Bombyx mori may be subjectedto sericin extraction, spun into yarns then used to create a matrix withhigh tensile strength suitable for applications such as bioengineeredligaments and tendons. Use of regenerated silk materials has also beenproposed for a number of medical purposes including wound protection,cell culture substrate, enzyme immobilization, soft contact lenses, anddrug-release agents.

Silk fibroin devices whether native, dissolved, or reconstituted, do nottypically contain cell-binding domains such as those found in collagen,fibronectin, and many other extracellular matrix (ECM) molecules.Fibroin is also strongly hydrophobic due to the β-sheet-rich crystallinenetwork of the core fibroin protein. These two factors couple toseverely limit the capacity of native host cells to bind to and interactwith implanted silk devices, as neither inflammatory cells likemacrophages or reparative cells like fibroblasts are able to attachstrongly, infiltrate and bioresorb the silk fibroin devices. In the caseof virgin silk and black braided (wax or silicone coated) silk sutures,this is typically manifested in a harsh foreign-body response featuringperipheral encapsulation. Substantially sericin-free silk experiences asimilar, though substantially fess vigorous response when implanted. Inessence, the host cells identify silk as a foreign body and opt to wallit off rather than interact with it. This severely limits the subsequentlong-term potential of the device particularly relating to tissuein-growth and remodeling and potentially, the overall utility of thedevice. If it is possible to provide a more effective biomaterialformulation for mediating host-device interactions whereby cells areprovided with a recognizable, acceptable and hence biocompatiblesurface, the biological, medicinal and surgical utility of silk isdramatically improved.

One possible means of introducing this improved cell-materialinteraction is to alter the silk fibroin material format into a morebiocompatible matrix. Manipulating the silk fibroin to make it into asilk hydrogel formulation is one particularly intriguing option becauseit consists of a silk protein network which is fully saturated withwater, coupling the molecular resiliency of silk with thebiocompatibility of a “wet” material. Generation of a silk hydrogel maybe accomplished in short by breaking apart native silk fibroin polymersinto its individual monomeric components using a solvent species,replacing the solvent with water, then inducing a combination of inter-and intra-molecular aggregation. It has been shown that the sol-geltransition can be selectively initiated by changing the concentration ofthe protein, temperature, pH and additive (e.g., ions and hygroscopicpolymers such as poly(ethylene oxide) (PEO), poloxamer, and glycerol).Increasing the silk concentration and temperature may alter the timetaken for silk gelation by increasing the frequency of molecularinteractions, increasing the chances of polymer nucleation. Anothermeans of accelerating silk gelation is through use of calcium ions whichmay interact with the hydrophilic blocks at the ends of silk moleculesin solution prior to gelation. Decreasing pH and the addition of ahydrophilic polymer have been shown to enhance gelation, possibly bydecreasing repulsion between individual silk molecules in solution andsubsequently competing with silk fibroin molecules in solution for boundwater, causing fibroin precipitation and aggregation.

Other silk fibroin gels have been produced by, for example, mixing anaqueous silk fibroin solution with protein derived biomaterials such asgelatin or chitosan. Recombinant proteins materials based on silkfibroin's structure have also been used to create self-assemblinghydrogel structures. Another silk gel, a silk fibroin-poly-(vinylalcohol) gel was created by freeze- or air-drying an aqueous solution,then reconstituting in water and allowing to self-assemble. Silkhydrogels have also been generated by either exposing the silk solutionto temperature condition of 4° C. (Thermgel) or by adding thirty percent(v/v) glycerol (Glygel). Silk hydrogels created via a freeze-thawprocess have not only been generated but also used in vitro as a cellculture scaffold.

The use of silk hydrogels as biomaterial matrices has also been exploredin a number of ways. General research on hydrogels as platforms for drugdelivery, specifically the release behavior of benfotiamine (a syntheticvariant of vitamin B₁) coupled to silk hydrogel was investigated. Thestudy revealed both silk concentration and addition of other compoundsmay factor in to the eventual release profile of the material.Similarly, the release of FITC-labeled dextran from a silk hydrogelcould be manipulated by altering the silk concentrations within the gel.

Further studies of silk hydrogels have been performed in vivo as well.For example, the material has been used in vivo to provide scaffoldingfor repair of broken bones in rabbits and showed an accelerated healingrate relative to control animals. Of particular interest, the in situstudy also illustrated that the particular formulation of silk hydrogeldid not elicit an extensive immune response from the host.

Despite early promise with silk hydrogel formulations in vivo, sericincontamination remains a concern in their generation and use just as withnative fibroin for reasons of biocompatibility as well as the potentialfor sericin to alter gelation kinetics. The existence of sericinmolecules in the silk solution intermediate prior to gelation may alsocompromise final gel structural quality, i.e., the distribution ofβ-sheet structure. For these reasons the removal of sericin from silkfibroin material prior to hydrogel manufacture remains a concern. Thepotential for disruption of gelation kinetics and structure bycontaminants also presents the need for development of a process whichconsistently ensures structural uniformity and biocompatibility.

SUMMARY

The present specification provides novel dermal fillers useful fortreating skin conditions.

Thus, aspects of the present specification disclose a compositioncomprising a gel phase, wherein the gel phase includes hydrogelparticles comprising a silk fibroin. In other aspects of the presentspecification disclose a composition comprising a gel phase and acarrier phase, wherein the gel phase includes hydrogel particlescomprising a silk fibroin.

Other aspects of the present specification disclose a compositioncomprising a gel phase, wherein the gel phase includes a) hydrogelparticles comprising a silk fibroin and b) hydrogel particles comprisinga matrix polymer. Matrix polymers useful to make such compositionsinclude, without limitation, glycosaminoglycans (like chondroitinsulfate, dermatan sulfate, keratan sulfate, heperan, heperan sulfate,and hyaluronan), lubricins, and polysaccharides (like dextran, dextrin,starch, hetastarch, glycogen, polyvinyl acetate, polyvinyl pyrrolidone,polyethylene glycol, polyethylene imine, cellulose, methyl cellulose,carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropylcellulose, hydroxyethyl methyl cellulose, and hydroxypropyl methylcellulose).

Yet other aspects of the present specification provide a method oftreating a skin condition in an individual in need thereof, the methodcomprising the steps of administering a composition disclosed hereininto a dermal region of the individual, wherein the administrationimproves the skin condition. Skin conditions treated by the disclosedcompositions include, without limitation, augmentations,reconstructions, diseases, disorders, defects, or imperfections of abody part, region or area. In one aspect, a skin condition treated bythe disclosed compositions include, without limitation, a facialaugmentation, a facial reconstruction, a facial disease, a facialdisorder, a facial defect, or a facial imperfection. In one aspect, askin condition treated by the disclosed compositions include, withoutlimitation, skin dehydration, a lack of skin elasticity, skin roughness,a lack of skin tautness, a skin stretch line or mark, skin paleness, adermal divot, a sunken check, a thin lip, a retro-orbital defect, afacial fold, or a wrinkle.

DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the impact of 23RGD on the gelation times of silkhydrogels manufactured under various circumstances for example withoutenhancers or with a water/23RGD enhancer (FIG. 1A), or with an ethanolenhancer or combined ethanol-23RGD enhancers (FIG. 1B). Depending uponthe ratio of 23RGD to silk used and the specific enhancer solvents, thepeptide may function as either an accelerant or decelerant of theprocess.

FIG. 2 is a graph of HPLC data illustrating the integration of 23RGD andstability of its binding to 4% silk gel material made with an enhancersolution consisting of a 3:1 molar ratio of 23RGD:silk dissolved in 90%ethanol, 10% water when rinsed multiple times in ultra-purified waterover several days. Data are shown for both total peak area andcalculated 23RGD:silk molar ratio based on a 23RGD standard curve.

FIG. 3 is a graph comparing gel dry mass component at different RGDconcentrations for 2% silk gels (A), 4% silk gels (B), and 6% gels (C).*Samples differ significantly, p<0.05; † sample differs significantlyfrom all others; ‡ all samples differ significantly.

FIG. 4 illustrates the impact upon silk hydrogel water absorption andretention as identified in a gel drying assay. Data are shown as thepercentage of mass retained by a silk gel sample (n=4 for each type)after being subjected to a 96-hour lyophilization process. Increasingconcentrations of 23RGD enhancer caused increasing dry mass in the gelmaterials more substantial than the mass of the peptide itself. Thisphenomenon is likely due to structural differences in 23RGD-enhancedgels which do not permit a level of water entrainment equal to those ofgels enhanced only with ethanol.

FIG. 5 shows a comparison of the percent mass loss over time due tobioresorption of samples cast by PG and EEG methods (A), cast fromincreasing silk concentrations (B), and cast using increasing RGDconcentrations (C). *Samples differ significantly, p<0.05; † samplediffers significantly from all others; ‡ all samples differsignificantly.

FIG. 6 illustrates wet mass loss due to proteolytic bioresorption ofsilk hydrogels enhanced by a combination of 23RGD and ethanol atincreasing concentrations of 23RGD. As a general trend, gels enhancedwith 23RGD tend to be bioresorbed more quickly based upon this assay.

FIG. 7 is a second illustration of the bioresorption behavior of23RGD-enhanced and non-23RGD-enhanced silk hydrogels when incubated in aprotease solution. This bioresorption data serves to reinforce thetrend, illustrated in FIG. 5, of a slightly more rapid rate ofbioresorption of 23RGD-enhanced hydrogels in comparison tonon-23RGD-enhanced gels. The figure also supports the more thoroughremoval of a-helix and random coil conformations from 23RGD-enhancedgels in FIG. 6 over four days of incubation in protease.

FIG. 8 shows structural features observed by Fourier-Transform Infrared(FTIR) spectroscopy of 4% silk fibroin hydrogel devices which areenhanced by ethanol alone, and two 23RGD-ethanol enhancers. The fullspectra (FIG. 9A) of the materials are compared and the Amide I Band(1700-1600 cm⁻¹) highlighted for particular attention (FIG. 9B) becauseof its relevance to secondary protein structure. Of specific interest isthe commonality between all gels in their rich β-sheet structure (1700cm⁻¹ and 1622 cm⁻¹ respectively, highlighted in FIGS. 9C and 9E) at alltime points. These peaks become more pronounced after bioresorption, andbegin to differentiate 23RGD-enhanced materials from materials enhancedwith ethanol alone. This is evidenced in 23RGD-enhanced gels by a peakshift to lower wave numbers by the 1622 cm⁻¹ peak and dramaticallyincreased prominence of the 1700 cm⁻¹ peak. Additional differencesbetween bioresorbed and non-bioresorbed gels may be seen in regions ofthe spectrum known to correlate to α-helix and random coil conformations(1654 cm⁻¹ and 1645 cm⁻¹ respectively highlighted in FIG. 9D). Theseconformations are extensively digested in all gel types, but mostcompletely in gels enhanced by 23RGD. This suggests that 23RGD-enhancedgels tend to bioresorb to a very β-sheet rich secondary structure in amore rapid fashion than non-23RGD-enhanced gels. Spectra shown werecollected on a Bruker Equinox 55 FTIR unit using a compilation of 128scans with a resolution of 4 cm⁻¹.

FIG. 9 shows a comparative FTIR spectra illustrating the effects ofdiffering gelation techniques on gel protein structure before (Day 0)and after (Day 4) proteolytic bioresorption. Groups assessed includedsamples cast by PG and EEG methods (A), cast from increasing silkconcentrations (B), and cast using increasing RGD concentrations (C).

FIG. 10 shows representative micrographs of H&E-stained histologicalsections collected from silk gels implanted subcutaneously in rats.Samples of 4% silk fibroin hydrogel formed by passive gelation (4P), 4%silk fibroin hydrogel formed by ethanol-enhanced gelation (4E), and 6%silk fibroin hydrogel formed by ethanol-enhanced gelation (6E) werecompared at 7 days (A, B, and E respectively) with 4E and 6E samplescompared again at days 28 (C and F) and 57 (D and G).

FIG. 11 shows representative gross photographs of 8% silk fibroinhydrogel devices both unmodified (A) and 23RGD-enhanced (D) after atwo-week subcutaneous incubation in Lewis rats. Also shown aremicrographs resultant from H & E stains of the unmodified (B and C) and23RGD-coupled (E and F) samples at 10× and 20× magnification. Thesegross images coupled with the histological micrographs provide evidenceof a less extensive inflammatory response during early deviceintegration being associated with 23RGD-enhanced gel thannon-23RGD-enhanced gel.

FIG. 12 shows representative histology collected from a thirteen-weekstudy of 4% 3:1 23RGD-enhanced silk hydrogel blended with 25% saline(left panels, H&E stain Trichrome stain) and ZYPLAST™ (right panels H&Estain, Trichrome stain) and injected into the intradermis of guinea pig.Each material type exhibited some clear evidence of implanted device in75% of their respective implant sites. These micrographs indicate strongsimilarities not only between the long-term bioresorptioncharacteristics but also long-term host tissue response betweencollagen-derived biomaterials and this particular 23RGD-enhanced silkhydrogel formulation.

FIG. 13 shows representative micrographs of H&E-stained histologicalsections collected from Day 28 explants of 4% silk fibroin, 10% saline(A); 4% silk fibroin, 1:1 23RGD, 10% saline (B); 6% silk fibroin, 1:123RGD, 10% saline (C); ZYPLAST™ (D); 4% silk fibroin, 25% saline (E); 4%silk fibroin, 1:1 23RGD, 25% saline (F); 6% silk fibroin, 10% saline(G); HYLAFORM™ (H); 6% silk fibroin, 25% saline (I); 4% silk fibroin,3:1 23RGD, 25% saline (J); and 6% silk fibroin, 1:1 23RGD, 25% saline(K).

FIG. 14 shows representative micrographs of Day 92 histological sectionsof 4% silk fibroin, 3:1 23RGD, 25% saline (A-D) and ZYPLAST™ samples(E-H) stained with H&E at 4× (A and E), 10× (B and F), stained withMasson's Trichrome at 10× (C and G) and under polarized light at 10× (Dand H).

FIG. 15 is a photograph of a custom-built testing jig used inconjunction with an Instron 8511 (Instron Corporation, Canton Mass.) inconjunction with Series IX software and a 100 N load cell forcharacterizing the injection forces associated with forcing silk gelthrough a 30 g needle.

FIG. 16 illustrates the average extrusion force data from mechanicaltesting of various silk gel formulations illustrating the effects ofchanging comminution method (A), saline concentration (B), silkconcentration (C), and RGD content (D). Values are reported as anaverage of n=4 tests at each displacement rate with standard deviationillustrated as error bars. *Samples differ significantly, p<0.05; †sample differs significantly from all others in group at same strainrate; ‡ all samples in group differ significantly from all others ingroup at same strain rate.

FIG. 17 shows representative ESEM micrographs of selectedRGD/ethanol-induced silk precipitates generated from the previouslymentioned formulations. BASE (A), SCVLO (B), RHI (C), RLO (D), AVHI (E),ECLO (F), AVLO (G), and 3R 6.7:1 (H) are shown at 200× magnification.

FIG. 18 shows a comparison of the total dry mass of precipitaterecovered from each silk precipitate formulation (n=4 for each type)after being subjected to a 96-hour lyophilization process. Data aregrouped to compare the effects of changing volume ratio of accelerantadded (A), concentration of 23RGD in the accelerant (B), changing theinitial silk concentration (C), and changing the concentration ofethanol in the accelerant (D). It was shown that increasing any of thesevolumes or concentrations resulted in greater quantities of precipitate,though none appear to have substantially greater impact than another.This phenomenon is likely due to basic kinetics of the assemblyreaction, with each reagent in turn appearing both as an excess and aslimiting dependent upon the specific formulation. *—significantdifference, p<0.05; †—Group differs significantly from all others.

FIG. 19 shows a comparison of the percentage of dry mass in each ofprecipitate recovered (n=4 for each type) after being subjected to a96-hour lyophilization process. Data are grouped to compare the effectsof changing volume ratio of accelerant added (A), concentration of 23RGDin the accelerant (B), changing the initial silk concentration (C), andchanging the concentration of ethanol in the accelerant (D). Increasingthe concentration of 23RGD used increased the dry mass percentage ofprecipitates, while increasing the ethanol percentage in the accelerantdecreased dry mass. These changes may stem from formation of altered gelnetwork structures caused by manipulation of these variables, likelymore crystalline in the case of 23RGD increases and less crystalline inthe case of ethanol concentration increases. *—significant difference,p<0.05; †—Group differs significantly from all others.

FIG. 20 shows representative FTIR spectra of the Amide I band for23RGD/ethanol-induced silk precipitates immediately after processing(D0). Spectra are grouped to compare the effects of changing volumeratio of accelerant added (A), concentration of 23RGD in the accelerant(B), changing the initial silk concentration (C), and changing theconcentration of ethanol in the accelerant (D). These spectra illustratethat similarities exist between all groups although changing 23RGDconcentrations and ethanol concentrations may substantially impactprecipitate structure. Increasing concentrations of decreased β-sheetseen in a peak shift from ˜1621 cm⁻¹ in RVLO to ˜1624 cm⁻¹ in RLO. Afurther increase in 23RGD concentration in both BASE and RHI caused thisweakened β-sheet again along with increased signal values in the 1654cm⁻¹ and 1645 cm⁻¹ range, correlating to increased random coil andα-helical content. An increased percentage of ethanol decreased thecontent of α-helical and random coil shown by decreased signal between1670 cm⁻¹ and 1630 cm⁻¹ in both ECLO and BASE samples relative to ECVLO.This decrease in a-helical and random coil is accompanied by an increasein β-sheet structure. The findings relating to 23RGD and ethanolconcentrations reinforce the trends observed in the percent dry mass ofthe precipitates, supposing that a-helical and random coil motifsentrain more water than β-sheet regions.

FIG. 21 is a representative micrograph of Congo red-stained23RGD/ethanol-induced silk precipitates under polarized light at 20×magnification. A lack of emerald-green birefringence indicates anegative result in testing for amyloid fibril formation.

FIG. 22 shows comparison of 23RGD:silk molar ratio in each ofprecipitate recovered. Data are grouped to compare the effects ofchanging volume ratio of accelerant added (A), concentration of 23RGD inthe accelerant (B), changing the initial silk concentration (C), andchanging the concentration of ethanol in the accelerant (D). Inexamining the 23RGD bound to the precipitates, all materials containedmore 23RGD than predicted by initial calculations aside of AVHI, RVLO,RHI, and SCVLO. In the cases of AVHI and ECLO the 23RGD quantity wassubstantially more than was expected. In the cases of BASE, RLO, SCVLO,and SCLO the 23RGD quantities were approximately double that expected.This may be indicative of the formation of a 23RGD dimer in the 90%ethanol accelerant solution. The RVLO samples were made with a 23RGDconcentration of 0.49 mg/mL in the accelerant, the lowest used in thisstudy and potentially within the solubility range of 23RGD in 90%ethanol. RLO samples used 1.47 mg/mL and most other formulations weremade with a 23RGD accelerant concentration of 2.45 mg/mL, above the23RGD concentration at which dimerization became favorable in thesolution. Further highlighting the possibility of 23RGD dimerizing inthe ethanol solution is the behavior of ECLO precipitation. The 23RGDconcentration remains 2.45 mg/mL as with BASE and AVLO but the waterconcentration in the accelerant is increased to 20% and results in abinding of about 1.5-fold the expected total of 23RGD instead of 2-fold.This may be due to dis-solution of a greater quantity of 23RGD, causingcoexistence between dimeric and monomeric 23RGD in solution reflected inthe subsequent binding ratios. *—significant difference, p<0.05; †—Groupdiffers significantly from all others; ‡—All groups differsignificantly.

FIG. 23 shows a representative FTIR spectra of the Amide I band areshown for 23RGD/ethanol-induced silk precipitates initially (D0) andafter proteolytic bioresorption (D2). Spectra are grouped to compare theeffects of changing volume ratio of accelerant added (A), concentrationof 23RGD in the accelerant (B), changing the initial silk concentration(C), and changing the concentration of ethanol in the accelerant (D).Accelerant quantity added did not substantially affect the bioresorptionbehavior of the materials as BASE, AVHI and AVLO all featured decreasedlevels of a-helix and random coil motifs. This decrease was slightlylarger in the case of AVLO which also featured a peak shift from 1624cm⁻¹ to 1622 cm⁻¹, indicating a more stable β-sheet structure. 23RGDconcentration did not appear to affect bioresorption behavior of thematerials either as RVLO, RLO, BASE and RHI all showed decreased inα-helix and random coil motifs, though a greater portion of a-helix andrandom coil remained intact in RHI. Silk concentration did notsubstantially affect the bioresorption behavior of the materials as BASEand SCLO exhibited decreased levels of a-helix and random coil motifsand featured slight peak shifts from 1624 cm⁻¹ to 1623 cm⁻¹.

FIG. 24 shows a H&E staining of tissue samples injected with a dermalfiller comprising 100% hyaluronan, a dermal filler comprising 95%hyaluronan and 5% silk fibronin hydrogel, a dermal filler comprising 75%hyaluronan and 25% silk fibronin hydrogel, a dermal filler comprising50% hyaluronan and 50% silk fibronin hydrogel, a dermal fillercomprising 25% hyaluronan and 75% silk fibronin hydrogel, and a dermalfiller comprising 100% silk fibronin hydrogel.

FIG. 25 shows on the tope row, a H&E staining of tissue samples injectedwith a dermal filler comprising 100% hyaluronan, a dermal fillercomprising 50% hyaluronan and 50% silk fibronin hydrogel, and a dermalfiller comprising 100% silk fibronin hydrogel; and on the bottom row aCD-68 staining of tissue samples injected with a dermal fillercomprising 100% hyaluronan, a dermal filler comprising 50% hyaluronanand 50% silk fibronin hydrogel, and a dermal filler comprising 100% silkfibronin hydrogel.

DETAILED DESCRIPTION

Aspects of the present specification provide, in part, a compositioncomprising a gel phase including a hydrogel comprising a silk fibroin.As used herein, the term “silk fibroin” is synonymous with “polymerizedsilk fibroin” and refers to silk fibroin existing primarily as apolymer. A hydrogel comprising polymerized silk fibroin or silk fibroinis made by, e.g., a gelation process disclosed herein.

Aspects of the present specification provide, in part, a depolymerizedsilk fibroin. As used herein, the term “depolymerized silk fibroin” issynonymous with “dissolved silk” and “dissolved silk fibroin” and refersto silk fibroin existing primarily as monomers or other lower oligomericunits. Treatment of naturally-occurring fibrous silk with a dissolutionagent, such as, e.g., a chaotropic agent results in depolymerized silkfibroin. The depolymerized silk fibroin used for preparing silk fibroinhydrogel is an intermediate in the silk hydrogel production process anda direct precursor to the hydrogel material. The depolymerized silkfibroin can be made from raw cocoons, previously degummed silk or anyother partially cleaned silk. This may also include material commonlytermed as “waste” from the reeling process, i.e. short fragments of rawor degummed silk, the sole precaution being that the silk must besubstantially cleaned of sericin prior to making fibroin solution andinducing gel formation. A particular source of raw silk is from commondomesticated silkworm B. marl, though several other sources of silk maybe appropriate. This includes other strains of Bombycidae includingAntheraea pernyi, Antheraea yamamai, Antheraea mylitta, Antheraeaassama, and Philosamia cynthia ricini,as well as silk producing membersof the families Saturnidae, Thaumetopoeidae, and silk-producing membersof the order Araneae. The material may also be obtained from otherspider, caterpillar, or recombinant sources.

A hydrogel disclosed herein provide for a depolymerized silk fibroinand/or silk fibroin that are substantially free of sericin. Methods forperforming sericin extraction have been described in pending U.S. patentapplication Ser. No. 10/008,924, U.S. Publication No. 2003/0100108,Matrix for the production of tissue engineered ligaments, tendons andother tissue. That application refers to cleaned fibroin fibers spuninto yarns, used to create a porous, elastic matrix suitable as asubstrate for applications requiring very high tensile strength, such asbioengineered ligaments and tendons.

Extractants such as urea solution, hot water, enzyme solutions includingpapain among others which are known in the art to remove sericin fromfibroin would also be acceptable for generation of the silk. Mechanicalmethods may also be used for the removal of sericin from silk fibroin.This includes but is not limited to ultrasound, abrasive scrubbing andfluid flow. The rinse post-extraction is conducted preferably withvigorous agitation to remove substantially any ionic contaminants,soluble, and in soluble debris present on the silk as monitored throughmicroscopy and solution electrochemical measurements. A criterion isthat the extractant predictably and repeatably remove the sericin coatof the source silk without significantly compromising the molecularstructure of the fibroin. For example, an extraction may be evaluatedfor sericin removal via mass loss, amino acid content analysis, andscanning electron microscopy. Fibroin degradation may in turn bemonitored by FTIR analysis, standard protein gel electrophoresis andscanning electron microscopy.

In certain cases, the silk utilized for generation of a silk hydrogelhas been substantially depleted of its native sericin content (i.e., s4% (w/w) residual sericin in the final extracted silk). Alternatively,higher concentrations of residual sericin may be left on the silkfollowing extraction or the extraction step may be omitted. In aspectsof this embodiment, the sericin-depleted silk fibroin has, e.g., about1% (w/w) residual sericin, about 2% (w/w) residual sericin, about 3%(w/w) residual sericin, or about 4% (w/w) residual sericin. In otheraspects of this embodiment, the sericin-depleted silk fibroin has, e.g.,at most 1% (w/w) residual sericin, at most 2% (w/w) residual sericin, atmost 3% (w/w) residual sericin, or at most 4% (w/w) residual sericin. Inyet other aspects of this embodiment, the sericin-depleted silk fibroinhas, e.g., about 1% (w/w) to about 2% (w/w) residual sericin, about 1%(w/w) to about 3% (w/w) residual sericin, or about 1% (w/w) to about 4%(w/w) residual sericin.

In certain cases, the silk utilized for generation of a silk hydrogel isentirely free of its native sericin content. As used herein, the term“entirely free (i.e. “consisting of” terminology) means that within thedetection range of the instrument or process being used, the substancecannot be detected or its presence cannot be confirmed.

In certain cases, the silk utilized for generation of a silk hydrogel isessentially free of its native sericin content. As used herein, the term“essentially free” (or “consisting essentially of”) means that onlytrace amounts of the substance can be detected.

Additionally, the possibility exists for deliberately modifying hydrogelproperties through controlled partial removal of silk sericin ordeliberate enrichment of source silk with sericin. This may function toimprove hydrogel hydrophilicity and eventual host acceptance inparticular biological settings despite sericin antigen icily.

After initial degumming or sericin removal from fibrous silk used forgeneration of a hydrogel, the silk is rinsed free of gross particulatedebris. It is of concern to remove such particles as either solvent(i.e., specific solvent of interest for device generation) soluble orinsoluble compounds may profoundly affect the outcome of the hydrogelgenerated from the intermediate solution. Insoluble compounds may serveas nucleation points, accelerating the gelation phenomenon andpotentially altering subsequent hydrogel protein structure. Solublecompounds may also serve to interface with the protein network of thehydrogel, altering the organizational state of the device. Either typeof compound could also compromise biocompatibility of the device.

Prior to dissolution, the prepared silk may be subjected to associationof various molecules. The binding between these compounds and the silkmolecules may be unaffected by the dissolving agent used for preparationof silk solution intermediate. The method for coupling the modifyingcompound to the prepared silk may vary dependent upon the specificnature of the bond desired between silk sequence and the modifier.Methods are not limited to but may include hydrogen bonding throughaffinity adsorption, covalent crosslinking of compounds or sequentialbinding of inactive and active compounds. These molecules may include,but would not be limited to, inorganic compounds, peptides, proteins,glycoproteins, proteoglycans, ionic compounds, natural, and syntheticpolymers. Such peptides, proteins, glycoproteins and proteoglycans mayinclude classes of molecules generally referred to as “growth factors”,“cytokines”, “chemokines”, and “extracellular matrix compounds”. Thesecompounds might include such things as surface receptor binding motifslike arginine-glycine-aspartic acid (RGD), growth factors like basicfibroblast growth factor (bFGF), platelet derived growth factor (PDGF),transforming growth factor (TGF), cytokines like tumor necrosis factor(TNF), interferon (IFN), interleukins (IL), and structural sequencesincluding collagen, elastin, hyaluronic acid and others. Additionallyrecombinant, synthetic, or non-native polymeric compounds might be usedas decoration including chitin, poly-lactic acid (PLA), andpoly-glycolic acid (PGA). Other compounds linked to the material mayinclude classes of molecules generally referred to as tracers,contrasting agents, aptamers, avimers, peptide nuclei acids and modifiedpolysaccharide coatings.

For example, the initially dissolved silk may be generated by a 4 hourdigestion at 60° C. of pure silk fibroin at a concentration of 200 g/Lin a 9.3 M aqueous solution of lithium bromide to a silk concentrationof 20% (w/v). This process may be conducted by other means provided thatthey deliver a similar degree of dissociation to that provided by a 4hour digestion at 60° C. of pure silk fibroin at a concentration of 200g/L in a 9.3 M aqueous solution of lithium bromide. The primary goal ofthis is to create uniformly and repeatably dissociated silk fibroinmolecules to ensure similar fibroin solution properties and,subsequently, device properties. Less substantially dissociated silksolution may have altered gelation kinetics resulting in differing finalgel properties. The degree of dissociation may be indicated byFourier-transform Infrared Spectroscopy (FTIR) or x-ray diffraction(XRD) and other modalities that quantitatively and qualitatively measureprotein structure. Additionally, one may confirm that heavy and lightchain domains of the silk fibroin dimer have remained intact followingsilk processing and dissolution. This may be achieved by methods such asstandard protein sodium-dodecyl-sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) which assess molecular weight of theindependent silk fibroin domains.

System parameters which may be modified in the initial dissolution ofsilk include but are not limited to solvent type, silk concentration,temperature, pressure, and addition of mechanical disruptive forces.Solvent types other than aqueous lithium bromide may include but are notlimited to aqueous solutions, alcohol solutions,1,1,1,3,3,3-hexafluoro-2-propanol, and hexafluoroacetone,1-butyl-3-methylimidazolium. These solvents may be further enhanced byaddition of urea or ionic species including lithium bromide, calciumchloride, lithium thiocyanate, zinc chloride, magnesium salts, sodiumthiocyanate, and other lithium and calcium halides would be useful forsuch an application. These solvents may also be modified throughadjustment of pH either by addition of acidic of basic compounds.

Further tailoring of the solvent system may be achieved throughmodification of the temperature and pressure of the solution, as idealdissolution conditions will vary by solvent selected and enhancersadded. Mechanical mixing methods employed may also vary by solvent typeand may vary from general agitation and mixing to ultrasonic disruptionof the protein aggregates. Additionally, the resultant dissolved silkconcentration may be tailored to range from about 1% (w/v) to about 30%(w/v). It may be possible to expand this range to include higherfractions of dissolved silk depending upon the specific solvent systemutilized. In one example, following initial dissolution of the processedsilk, the silk protein may be left in a pure aqueous solution at 8%(w/v) silk. This is accomplished by removal of the residual solventsystem while simultaneously ensuring that the aqueous component of thesilk solution is never fully removed nor compromised. In a situationwhich involves an initial solution of 200 g/L silk in a 9.3 M aqueoussolution of lithium bromide, this end is accomplished by a dialysisstep.

In aspects of this embodiment, the depolymerized silk fibroin (dissolvedsilk fibroin) has a concentration of, e.g., about 1% (w/v), about 2%(w/v), about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v),about 7% (w/v), about 8% (w/v), about 9% (w/v), about 10% (w/v), about12% (w/v), about 15% (w/v), about 18% (w/v), about 20% (w/v), about 25%(w/v), or about 30% (w/v). In other aspects of this embodiment, thedepolymerized silk fibroin (dissolved silk fibroin) has a concentrationof, e.g., at least 1% (w/v), at least 2% (w/v), at least 3% (w/v), atleast 4% (w/v), at least 5% (w/v), at least 6% (w/v), at least 7% (w/v),at least 8% (w/v), at least 9% (w/v), at least 10% (w/v), at least 12%(w/v), at least 15% (w/v), at least 18% (w/v), at least 20% (w/v), atleast 25% (w/v), or at least 30% (w/v). In yet other aspects of thisembodiment, the depolymerized silk fibroin (dissolved silk fibroin) hasa concentration of, e.g., about 1% (w/v) to about 5% (w/v), about 1%(w/v) to about 10% (w/v), about 1% (w/v) to about 15% (w/v), about 1%(w/v) to about 20% (w/v), about 1% (w/v) to about 25% (w/v), about 1%(w/v) to about 30% (w/v), about 5% (w/v) to about 10% (w/v), about 5%(w/v) to about 15% (w/v), about 5% (w/v) to about 20% (w/v), about 5%(w/v) to about 25% (w/v), about 5% (w/v) to about 30% (w/v), about 10%(w/v) to about 15% (w/v), about 10% (w/v) to about 20% (w/v), about 10%(w/v) to about 25% (w/v), or about 10% (w/v) to about 30% (w/v).

Example dialysis conditions include a 3 mL-12 mL sample volume dialysiscassettes with 3.5 kD molecular weight cutoff cellulose membranesdialyzed for three days against ultra-pure water with a series of sixchanges at regular intervals while stirring constantly. Each cassette, 5mL-12 mL cartridge size, may be loaded (for example via 20-mL syringe)with 12 mL of a 20% solution of silk dissolved in 9.3 M lithium bromidevia an 18 gauge needle. The resultant silk solution may be 8%±0.5%(w/v). The silk solution may be stored at a range of −80° C. to 37° C.,such as 4° C. prior to use. One method is to dialyze the solutionagainst water using a 3.5 kD molecular weight cutoff cellulose membrane,for example, at one 12 mL cartridge per 1 L water in a 4 L beaker withstirring for 48 hours or 72 hours. Water may be changed several timesduring the dialysis, for example at 1 hour, 4 hours, 12 hours, 24 hours,and 36 hours (total of six rinses). In other embodiments, this membranemay take the shape of a cassette, tubing or any other semi-permeablemembrane in a batch, semi-continuous or continuous system. If desired,the concentration of silk in solution may be raised following theoriginal dialysis step by inclusion of a second dialysis against ahygroscopic polymer such as PEG, a poly(ethylene oxide) or amylase.

The parameters applied to the dialysis step may be altered according tothe specific needs or requirements of the particular solution systeminvolved. Although it may be undesirable to change membrane compositionor pore size in the interests of maintaining efficiency of the process,it would be possible to change the structuring of the dialysis barrier,as a dialysis tube or any large semi-permeable membrane of similarconstruction should suffice. Additionally it should be considered thatany alteration in the nature of the physical dialysis interface betweensolution and buffer might alter rates of ion flux and thereby createmembrane-localized boundary conditions which could affect solutiondialysis and gelation rate kinetics. The duration and volume ratiosassociated with this dialysis process must be tailored to any new systemas well, and removal of the solvent phase should be ensured afterpurification before proceeding.

It is also possible to change the buffer phase in the dialysis system,altering water purity or adding hygroscopic polymers to simultaneouslyremove ions and water from the initial silk solution. For example, ifnecessary, the silk solution can be concentrated by dialysis against ahygroscopic polymer, for example, PEG, a polyethylene oxide or amylase.The apparatus used for dialysis can be cassettes, tubing, or any othersemi-permeable membrane.

Insoluble debris may be removed from the dialyzed silk solution bycentrifugation or filtration. For example, the dialyzed silk may beremoved from the cassette with a needle and syringe (e.g., an 18 gneedle at 20 mL syringe), and placed into a clean centrifuge tube withsufficient volume (e.g., 40 mL). The centrifuge may be run at 30,000 grelative centrifugal force (RCF) for 30 minutes at 4° C. The resultingsupernatant may be collected and centrifuged again under identicalconditions, and the remaining supernatant collected (e.g., in a 50 mLtest tube) and stored at 4° C. The silk solution may also be evaluatedvia X-ray photoelectron spectroscopy (to check for lithium bromideresidue) and dry mass (to check solution for dry protein mass,concentration w/v).

Additionally, dependent upon the initial silk solvent, it might bedesirable to remove portions of either the silk phase or solvent phasefrom the solution via an affinity column separation. This could beuseful in either selectively binding specific solvent molecules orspecific solute molecules to be eluted later in a new solvent. Thepossibility also exists for a lyophilization of the depolymerized silkfibroin (dissolved silk) followed by a reconstitution step. This wouldbe most useful in a case where removing a solvent, is unlikely to leaveresidue behind. In the case of a lyophilized solution, either used as apurification step or as a procedure subsequent to purification, the typeof solvent used for reconstitution might be tailored for the process athand. Desirable solvents might include but are not limited to aqueousalcohol solutions, aqueous solutions with altered pH, and variousorganic solutions. These solvents may be selected based upon a number ofparameters which may include but are not limited to an enhanced gelationrate, altered gel crystalline structure, altered solution intermediateshelf-life, altered silk solubility, and ability to interact withenvironmental milieu such as temperature and humidity.

In certain embodiments, a silk hydrogel is prepared from dissolved silkfibroin solution that uses an agent to enhance gelation and an agent toimprove the gel's biocompatibility. In some instances, the same agentboth enhances gelation and improves biocompatibility. An example agentthat both improves gel biocompatibility and serves as a gelationenhancer is an amphiphilic peptide which binds to silk molecules throughhydrophobic interactions, such as, e.g., a non-RDG integrin or a RGDmotif containing peptide like 23RGD. In other instances, differentagents serve these purposes. An example of an agent that serves as agelation enhancer is an alcohol, such as, e.g., ethanol, methanol, andisopropanol; glycerol; and acetone.

Regarding gelation enhancers, to accelerate the phenomenon of silkgelation, a depolymerized silk fibroin solution (dissolved silksolution) may be mixed with pure alcohol or aqueous alcohol solution atvaried volume ratios accompanied by mixing, either through stirring,shaking or any other form of agitation. This alcohol solution enhancermay then have a quantity of an amphiphilic peptide added as a furtherenhancer of the final gel outcome. The extent of acceleration may beheightened or lessened by adding a larger or smaller enhancer componentto the system.

In addition to organics, the gelation rate may be enhanced by increasingthe concentration of the depolymerized silk fibroin (dissolved silk).This is done by methods including but not limited to dialysis ofintermediate silk solution against a buffer incorporating a hygroscopicspecies such as polyethylene glycol, a lyophilization step, and anevaporation step. Increased temperature may also be used as an enhancerof the gelation process. In addition to this, manipulation ofintermediate silk solution pH by methods including but not limited todirect titration and gas exchange may be used to enhance the gelationprocess. Introduction of select ionic species including calcium andpotassium in particular may also be used to accelerate gelation rate.

Nucleating agents including organic and inorganic species, both solubleand insoluble in an aqueous silk solution intermediate may be used toenhance the gelation process. These may include but are not limited topeptide sequences which bind silk molecules, previously gelled silk, andpoorly soluble p-sheet rich structures. A further means of acceleratingthe gelation process is through the introduction of mechanicalexcitation. This might be imparted through a shearing device, ultrasounddevice, or mechanical mixer. It should be borne in mind that any ofthese factors might conceivably be used in concert with any other orgroup of others and that the regime would need to be tailored to thedesired outcome.

The time necessary for complete silk solution gelation may vary fromseconds to hours or days, depending on the values of the above mentionedparameters as well as the initial state of aggregation and organizationfound in the silk solution (FIG. 1). The volume fraction of addedenhancer may vary from about 0% to about 99% of the total system volume(i.e., either component may be added to a large excess of the other orin any relative concentration within the interval). The concentration ofsilk solution used can range from about 1% (w/v) to about 20% (w/v). Theenhancer can be added to silk solution or the silk solution can be addedto enhancer. The formed silk hydrogel may be further chemically orphysically cross-linked to gain altered mechanical properties.

In aspects of this embodiment, an enhancer solution is added to adepolymerized silk fibroin (dissolved silk fibroin) solution, thedepolymerized silk fibroin solution having a concentration ofdepolymerized silk fibroin of, e.g., about 1% (w/v), about 2% (w/v),about 3% (w/v), about 4% (w/v), about 5% (w/v), about 6% (w/v), about 7%(w/v), about 8% (w/v), about 9% (w/v), about 10% (w/v), about 12% (w/v),about 15% (w/v), about 18% (w/v), about 20% (w/v), about 25% (w/v), orabout 30% (w/v). In other aspects of this embodiment, an enhancersolution is added to a depolymerized silk fibroin (dissolved silkfibroin) solution, the depolymerized silk fibroin solution having aconcentration of depolymerized silk fibroin of, e.g., at least 1% (w/v),at least 2% (w/v), at least 3% (w/v), at least 4% (w/v), at least 5%(w/v), at least 6% (w/v), at least 7% (w/v), at least 8% (w/v), at least9% (w/v), at least 10% (w/v), at least 12% (w/v), at least 15% (w/v), atleast 18% (w/v), at least 20% (w/v), at least 25% (w/v), or at least 30%(w/v). In yet other aspects of this embodiment, an enhancer solution isadded to a depolymerized silk fibroin (dissolved silk fibroin) solution,the depolymerized silk fibroin solution having a concentration ofdepolymerized silk fibroin of, e.g., about 1% (w/v) to about 5% (w/v),about 1% (w/v) to about 10% (w/v), about 1% (w/v) to about 15% (w/v),about 1% (w/v) to about 20% (w/v), about 1% (w/v) to about 25% (w/v),about 1% (w/v) to about 30% (w/v), about 5% (w/v) to about 10% (w/v),about 5%(w/v) to about 15% (w/v), about 5%(w/v) to about 20% (w/v),about 5% (w/v) to about 25% (w/v), about 5% (w/v) to about 30% (w/v),about 10% (w/v) to about 15% (w/v), about 10% (w/v) to about 20% (w/v),about 10% (w/v) to about 25% (w/v), or about 10% (w/v) to about 30%(w/v).

Aspects of the present specification provide, in part, a hydrogelcomprising an amphiphilic peptide. As used herein, the term “amphiphilicpeptide” refers to a peptide that includes both hydrophobic andhydrophilic properties. Many other amphiphilic molecules interactstrongly with biological membranes by insertion of the hydrophobic partinto the lipid membrane, while exposing the hydrophilic part to theaqueous environment. Particular embodiments of hydrogels include silkfibroin, silk fibroin with 23RGD, silk fibroin with alcohol and 23RGD,and silk fibroin with alcohol, 23RGD, and saline/PBS. The amount,relative ratio and sequence of adding the components will changeaccording to the specific requirement for the device.

Additionally, an amphiphilic peptide may accelerate the phenomenon ofsilk gelation under certain circumstances. Such gel may be producedthrough combination of dissolved silk fibroin solution and an enhancersolution of amphiphilic peptide in alcohol across the silk concentrationranges from about 1% (w/v) to about 20% (w/v), amphiphilic peptideconcentration ranges from about 1:100 to 100:1 moles 23RGD:moles silk,and alcohol concentration ranges from about 1% (v/v) to about 99% (v/v)before removal. Thus, for example, a particular silk gel is producedthrough direct contact between an aqueous solution of depolymerized silkfibroin and an enhancer solution comprising 23RGD in ethanol. Forexample, the dissolved silk solution may be mixed with a 23RGD suspendedin pure ethanol or aqueous ethanol solution at varied volume ratiosaccompanied by mixing, either through stirring, shaking or any otherform of agitation.

More specifically, as a non-limiting example, to infuse the silk fibroinhydrogel with 23RGD, the 23RGD is first dissolved in a solution ofethanol and water (e.g., 90% ethanol in purified water) in an amount togenerate the planned silk and 23RGD concentrations of the final gel, andmixed (e.g., vortexed until there is no visible 23RGD particulate). Thissolution is then mixed with dissolved silk solution (e.g., by pipettingrapidly for 1-2 seconds). The gelling mixture may be allowed to standcovered under ambient conditions for a suitable period, for example 24hours (or 24 hours after the gel has solidified depending on enhancerconditions).

The amount of time required for dissolved silk solutions to gel may varyfrom seconds to hours or days, depending on the ratio of silk solutionvolume and enhancer solution volume, dissolved silk fibroinconcentration, enhancer solution concentration, enhancer type andamphiphilic peptide concentration. The amphiphilic peptide may be mixedinto the dissolved silk solution in a variety of ways, for examplewater-dissolved amphiphilic peptide can be added to a dissolved silksolution to form a gel; an amphiphilic peptide can be added to water,blended with an alcohol, then added to a dissolved silk solution; or anamphiphilic peptide can be added to a silk fibroin hydrogel. The molarratio of amphiphilic peptide:silk fibroin can range from 100 to 0.01,the dissolved silk solution concentration can be from about 1% to about20%.

An example of an amphiphilic peptide is a 23RGD peptide having the aminoacid sequence:HOOC-Gly-Arg-Gly-Asp-Ile-Pro-Ala-Ser-Ser-Lys-Gly-Gly-Gly-Gly-Ser-Arg-Leu-Leu-Leu-Leu-Leu-Leu-Arg-NH₂(abbreviated HOOC-GRGDIPASSKG₄SRL₆R—NH₂) (SEQ ID NO: 1). Optionally,each of the arginine residues may be of the D-form, which may stabilizethe RG bond to serine proteases. Additionally, the COO-terminus may beacylated to block proteolysis. This example 23RGD has the amino acidsequence Ac-GdRGDIPASSKG₄SdRL_(6d)R—NH₂ (SEQ ID NO: 2). It may beadvantageous to include a spacer domain in the RGD peptide, for example,a peptide such as SG₄KSSAP (SEQ ID NO: 3) may present the RGD on thesurface of the silk biomaterial by optimally separating the cellattachment domain from the bonding sequence at the end of the peptide.The optional leucine tails of this example may interact in a fashionanalogous to a leucine zipper, and be driven by entropy from an aqueoussolution to form an approximation of a Langmuir-Blodgett (LB),monomolecular film on the surface of materials exposed to suchsolutions, thus presenting a ‘carpet’ of RGD attachment sites on thosesurfaces.

Other proteins or peptides may be used instead of 23RGD if such proteinsor peptides have the desired characteristics. Example characteristicsinclude hydrophilic domains that can interfere/enhance/affect silkgelation, and/or cell integrin binding domains that enhance celladhesion, spreading, and migration. Non-limiting examples of suchnon-RDG integrins include, KQAGDV (SEQ ID NO: 4), PHSRN (SEQ ID NO: 5),YIGSR (SEQ ID NO: 6), CDPGYIGSR (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8),RNIAEIIKDI (SEQ ID NO: 9), YFQRYLI (SEQ ID NO: 10), PDSGR (SEQ ID NO:11), FHRRIKA (SEQ ID NO: 12), PRRARV (SEQ ID NO: 13), and WQPPRARI (SEQID NO: 14). See also Hersel et al., 24 Biomaterials 4285-415 (2003).

In aspects of this embodiment, a hydrogel comprises a molar ratio ofamphiphilic peptide to silk fibroin of, e.g., about 100:1, about 90:1,about 80:1, about 70:1, about 60:1, about 50:1, about 40:1, about 30:1,about 20:1, about 10:1, about 7:1, about 5:1, about 3:1, about 1:1,about 1:3, about 1:5, about 1:7, about 1:10, about 1:20, about 1:30,about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, or about1:90, or about 1:100. In other aspects of this embodiment, a hydrogelcomprises a molar ratio of amphiphilic peptide to silk fibroin of, e.g.,at least 100:1, at least 90:1, at least 80:1, at least 70:1, at least60:1, at least 50:1, at least 40:1, at least 30:1, at least 20:1, atleast 10:1, at least 7:1, at least 5:1, at least 3:1, at least 1:1, atleast 1:3, at least 1:5, at least 1:7, at least 1:10, at least 1:20, atleast 1:30, at least 1:40, at least 1:50, at least 1:60, at least 1:70,at least 1:80, or at least 1:90, or at least 1:100. In yet other aspectsof this embodiment, a hydrogel comprises a molar ratio of amphiphilicpeptide to silk fibroin of, e.g., at most 100:1, at most 90:1, at most80:1, at most 70:1, at most 60:1, at most 50:1, at most 40:1, at most30:1, at most 20:1, at most 10:1, at most 7:1, at most 5:1, at most 3:1,at most 1:1, at most 1:3, at most 1:5, at most 1:7, at most 1:10, atmost 1:20, at most 1:30, at most 1:40, at most 1:50, at most 1:60, atmost 1:70, at most 1:80, or at most 1:90, or at most 1:100. In stillother aspects of this embodiment, a hydrogel comprises a molar ratio ofamphiphilic peptide to silk fibroin of, e.g., about 100:1 to about1:100; about 90:1 to about 1:90; about 80:1 to about 1:80; about 70:1 toabout 1:70; about 60:1 to about 1:60; about 50:1 to about 1:50; about40:1 to about 1:40; about 30:1 to about 1:30; about 20:1 to about 1:20;about 10:1 to about 1:10; about 7:1 to about 1:7; about 5:1 to about1:5; or about 3:1 to about 1:3.

The use of an amphiphilic peptide not only alters the protein structurecharacteristics of silk fibroin protein, but in so doing alters itsresistance to proteolytic bioresorption in vitro. These alterations inproteolytic bioresorption resistance stem from aspects of the proteinstructure alteration as α-helix and random coil are typically thought tobe less stable and therefore more susceptible to proteolyticbioresorption than β-sheet regions of silk. β-turn and β-strand regionsof the hydrogel disclosed herein are most resistant to proteolyticbioresorption as opposed to regions of α-helixes and random coils.Through deliberate manipulation of this protein structure by means ofcontrolled solution concentration and addition of enhancer factors(type, concentration, and driving gradient), gelation kinetics andresultant gel properties might be controlled to deliver optimal outcomesin terms of degradative and resultant biological behaviors. The impactof amphiphilic peptide addition to silk hydrogel in a silk hydrogel isevident upon examination of data obtained through implantation studiesconducted in vivo, both subcutaneously in rats and intradermally in thedermis of guinea pigs. See Example 10.

Aspects of the present specification provide, in part, a hydrogelcomprising a five-amino acid peptide “tail” capable of linking orconjugating a molecule X to a silk molecule or fibroin when the moleculeX is attached to the tail. A molecule X is any entity, natural orsynthetic, that can be useful and can be use in the context of silkhydrogels. As used herein, the term “linking” or “conjugating” in thecontext of molecule X refers to an indirect physical attachment of amolecule X to a silk fibroin via a third entity, the five-amino acidpeptide “tail” being that entity. In one embodiment, the tail binds tosilk fibroin by hydrophobic interaction to the silk fibroin.Alternatively, the “tail” binds the silk molecules by hydrogen bondingand/or covalen_(t) bonding. It is envisioned that the “tail” can bindsilk fibroins by a combination of hydrophobic interactions, hydrogenbonds, and covalent bonds. By attaching a molecule X to a “tail”described herein, it is possible to indirectly link the molecule X tosilk fibroin via the tail, and thus to the silk hydrogels describedherein.

In one embodiment, the molecule X is attached to a tail at the carboxyl(COOH) end of the five-amino acid peptide. In another embodiment, themolecule X is attached to a tail at the amino (NH₂) end of thefive-amino acid peptide.

In one embodiment, the five-amino acid peptide “tail” compriseshydrophobic and/or apolar (non polar) amino acid residues such asvaline, leucine, isoleucine, phenylalanine, tryptophan, methionine,cysteine, alanine, tyrosine, serine, proline, histidine, threonine andglycine. Various combinations of hydrophobic and/or apolar amino acidresidues are possible, for e. g. LLLLL (SEQ ID NO: 15), LLFFL (SEQ IDNO: 16), LFLWL (SEQ ID NO: 17), FLWLL (SEQ ID NO: 18) and LALGL (SEQ IDNO: 19). In other embodiments, the tail comprises any combination of thetwenty standard conventional amino acid residues. In other embodiments,the tail comprises hydrophobic and/or apolar (non polar) and amino acidsresidues with hydrophobic side chains, e.g. arginine and lysine. As usedherein, the term “comprising” or “comprises” means that other elementscan also be present in addition to the defined elements presented. Theuse of “comprising” indicates inclusion rather than limitation.

In one embodiment, the five-amino acid peptide “tail” capable of linkingor conjugating a molecule X to a silk molecule or fibroin when themolecule X is attached to the tail comprise more than five amino acidresidues, e. g. six or seven hydrophobic and/or apolar amino acidresidues, such as LLLLLL (SEQ ID NO: 20).

In one embodiment, the five-amino acid peptide “tail” comprises aminoacid residues that are part hydrophobic (i.e. the part of the side-chainnearest to the protein main-chain), for e.g. arginine and lysine. in oneembodiment, the part hydrophobic amino acid residues flank thefive-amino acid peptide “tail” such as in RLLLLLR (SEQ ID NO: 21),KLLLLLR (SEQ ID NO: 22) and KLLLLLK (SEQ ID NO: 23).

In one embodiment, the five-amino acid peptide “tail” is separated froma molecule X by a spacer peptide. Spacer peptides should generally havenon-polar amino acid residues, such as, glycine and proline. In oneembodiment, the spacer comprises unnatural amino acid residues such asnor amino acids and keto-substituted amino acids. Such unnatural aminoacid residues are well known to one skilled in the art. In oneembodiment, the spacer peptide is attached to a tail at the carboxyl(COOH) end of the five-amino acid peptide. In another embodiment, thespacer is attached to a tail at the amino (NH₂) end of the five-aminoacid peptide.

The length of the space peptide is variable. The spacer serves to linkthe molecule X and tail together and also to provide steric freedom tothe molecule X, allowing for proper orientation of a molecule X (e. g.cell binding domains such as the RGD domain) and the correct interactionof the molecule X with cells in vivo. A spacer which is too short canprevent the molecule X from being properly functional (i.e., holding ittoo tight to the silk molecules and away from cells), a spacer which istoo long can cause undesired effects as well (i.e., non-specificassociation of peptides or shortened efficacy from peptide due to spacerbreakage). In one embodiment, the number of amino acid residues in aspacer can range form 1 to 300. In one embodiment, the spacer comprisesa single amino acid residue, such as a G or a P. Examples of spacerswith more amino acid residues are GSPGISGGGGGILE (SEQ ID NO: 24) andSGGGGKSSAPI (SEQ ID NO: 25).

In one embodiment, the molecule X is any biological molecule or fragmentthereof. Examples biological molecules include but are not limited togrowth factors, hormones, cytokines, chemokines, extracellular matrixcompounds, osteogenic protein (OP), bone morphogenetic protein (BMP),growth and differentiation factor (GDF), transforming growth factor(TGF), epidermal growth factor (EGF), vascular endothelial growth factor(VEGF), interleukin (IL), platelet derived growth factor (PDGF),fibroblast growth factor (FGF), insulin-like growth factor (IGF), basicfibroblast growth factor (BFGF), fibroblast activation protein (FAP),disintegrin, metalloproteinase (ADAM), matrix metalloproteinase (MMP),connective tissue growth factor (CTGF), stromal derived growth factor(SDGF), keratinocyte growth factor (KGF), tumor necrosis factor (TNF),interferon (IFN), erythropoietin (EPO), hepatocyte growth factor (HGF),thrombopoietin (TPO), granulocyte colony stimulating factor (GCSF),granulocyte macrophage colony stimulating factor (GMCSF), myostatin(GDF-8), collagen, resilin, elastin, laminin, hyaluronic acid, decorin,actin, and tubulin. Examples fragments of biological molecules includebut are not limited to known cell integrin binding domains including butnot limited to RGD, KQAGDV (SEQ ID NO: 4), PHSRN (SEQ ID NO: 5), YIGSR(SEQ ID NO: 6), CDPGYIGSR (SEQ ID NO: 7), IKVAV (SEQ ID NO: 8),RNIAEIIKDI (SEQ ID NO: 9), YFQRYLI (SEQ ID NO: 10), PDSGR (SEQ ID NO:11), FHRRIKA (SEQ ID NO: 12), PRRARV (SEQ ID NO: 13), and WQPPRARI (SEQID NO: 14).

In other embodiments, the molecule X is any recombinant, synthetic, ornon-native polymeric compounds. Examples include but are not limited tochitin, poly-lactic acid (PLA), poly-glycolic acid (PGA), as tracers (e.g. radioisotopes), contrasting agents (e. g. imaging dyes), aptamers,avimers, peptides, nuclei acids, modified polysaccharide coatings, drugs(chemotherapy drugs),and recombinant antibodies or antibody-basedmoieties.

In one embodiment, the present specification provides a syntheticmolecule having the formula: (molecule X)_(n)-(spacerpeptide)₀₋₃₀₀-(tail)-NH₂ for linking with silk molecule or fibroin,wherein “n” is a whole integer ranging from 1-30, and wherein the aminoacid residues of the spacer ranges from 0-300. Examples of suchsynthetic molecule capable for linking to silk molecule or fibroin are:GRGDIPASSKG₄SRL₆R—NH₂ (SEQ ID NO: 1), Ac-GdRGDIPASSKG₄SdRL₆dR—NH₂ (SEQID NO: 2), (VEGF)-(VEGF)-GSPGISGGGGGILEKLLLLLK—NH₂ (SEQ ID NO: 26),(HIV-C-peptide)₃-GSPGISGGGGGILEKLALWLLR-NH₂ (SEQ ID NO: 27),(taxol)₂-GSPGISGGGGGILERLLLLR—NH₂ (SEQ ID NO: 28), and(EPO)₂-GSPGISGGGGGILERLLWLLR—NH₂ (SEQ ID NO: 29). When used in thecontext of the silk hydrogel described herein, the synthetic molecule ofSEQ ID NO: 1 enable better tissue attachment of the hydrogel constructin vivo, the synthetic molecule of SEQ ID NO: 26 can promote bloodvessel generation (neo-angiogenesis) in tissue engineered constructs,the synthetic molecule of SEQ ID NO: 28 can provide a slow releaseanti-HIV medication in the form of a transdermal delivery patch, thesynthetic molecule of SEQ ID. NO: 28 can provide sustained dosage ofanti-cancer drug in vivo, and the synthetic molecule of SEQ ID NO: 29can provide a slow release EPO during cancer chemotherapy treatment.

Aspects of the present specification disclose, in part, a hydrogelcomprising a synthetic molecule having the formula: (moleculeX)_(n)-(spacer peptide)₀₋₃₀₀-(tail)-NH₂ or a synthetic molecule havingthe formula: (molecule X)_(n)-(spacer peptide)₀₋₃₀₀-(tail)-NH₂ and anamphiphilic peptide. In one embodiment, the amphiphilic peptide is23RGD. In one embodiment, the present specification provides a method ofconjugating a molecule X to a silk molecule or fibroin comprising mixinga synthetic molecule having the formula: (molecule X)_(n)-(spacerpeptide)₀₋₃₀₀-(tail)-NH₂ with a silk molecule or fibroin or silksolution. Conjugation of individual peptide can be effected by a linkagevia the N-terminal or the C-terminal of the peptide, resulting in anN-linked peptide oligomer or a C-linked peptide oligomer, respectively.

Methods of peptide synthesis are known to one skilled in the art, forexample, the peptides described herein can be synthetically constructedby suitable known peptide polymerization techniques, such as exclusivelysolid phase techniques, partial solid-phase techniques, fragmentcondensation or classical solution couplings. For example, the disclosedpeptides can be synthesized by the solid phase method using standardmethods based on either t-butyloxycarbonyl (BOC) or9-fluorenylmethoxy-carbonyl (FMOC) protecting groups. This methodologyis described by G. B. Fields et al. in Synthetic Peptides: A User'sGuide, W. M. Freeman & Company, New York, N.Y., pp. 77-183 (1992) and inthe textbook “Solid-Phase Synthesis”, Stewart & Young, Freemen &Company, San Francisco, 1969, and is exemplified by the disclosure ofU.S. Pat. No. 4,105,603, issued Aug. 8, 1979. Classical solutionsynthesis is described in detail in “Methoden der Organischen Chemic(Houben-Weyl): Synthese von Peptiden”, E. Wunsch (editor) (1974) GeorgThieme Verlag, Stuttgart West Germany. The fragment condensation methodof synthesis is exemplified in U.S. Pat. No. 3,972,859. Other availablesyntheses are exemplified in U.S., Pat. No. 3,842,067, U.S. Pat. No.3,872,925, issued Jan. 28, 1975, Merrifield B, Protein Science (1996),5: 1947-1951; The chemical synthesis of proteins; Mutter M, Int J PeptProtein Res 1979 March; 13 (3): 274-7 Studies on the coupling rates inliquid-phase peptide synthesis using competition experiments; and SolidPhase Peptide Synthesis in the series Methods in Enzymology (Fields, G.B. (1997) Solid-Phase Peptide Synthesis. Academic Press, San Diego.#9830).The foregoing disclosures are incorporated herein by reference.Molecular DNA methods can also be used. The coding sequence of the shortspacer can be constructed be annealing a complementary pair of primers.One of skill in the art can design and synthesize oligonucleotides thatwill code for the selected spacer.

Methods of linking peptides are also known in the art. The physicallinking of the individual isolated peptides into oligomeric peptides asset forth herein, can be effected by chemical conjugation procedureswell known in the art, such as by creating peptide linkages, use ofcondensation agents, and by employing well known bifunctionalcross-linking reagents. The conjugation may be direct, which includeslinkages not involving any intervening group, e.g., direct peptidelinkages, or indirect, wherein the linkage contains an interveningmoiety, such as a protein or peptide, e.g., plasma albumin, or otherspacer molecule. For example, the linkage may be via aheterobifunctional or homobifunctional cross-linker, e.g., carbodiimide,glutaraldehyde, N-succinimidyl 3-(2-pyridydithio) propionate (SPDP) andderivatives, bis-maleimide,4-(N-maleimidomethyl)cyclohexane-1-carboxylate, and the like.

Cross-linking can also be accomplished without exogenous cross-linkersby utilizing reactive groups on the molecules being conjugated. Methodsfor chemically cross-linking peptide molecules are generally known inthe art, and a number of hetero- and homobifunctional agents aredescribed in, e.g., U.S. Pat. Nos. 4,355,023, 4,657,853, 4,676,980,4,925,921, and 4,970,156, and Immuno Technology Catalogue and Handbook,Pierce Chemical Co. (1989), each of which is incorporated herein byreference. Such conjugation, including cross-linking, should beperformed so as not to substantially affect the desired function of thepeptide oligomer or entity conjugated thereto, including therapeuticagents, and moieties capable of binding substances of interest.

It will be apparent to one skilled in the art that alternative linkerscan be used to link peptides, for example the use of chemical proteincrosslinkers. For example homobifunctional crosslinker such asdisuccinimidyl-suberimidate-dihydrochloride;dimethyl-adipimidate-dihydrochloride; 1,5,-2, 4dinitrobenezene orheterobifunctional crosslinkers such as N-hydroxysuccinimidyl2,3-dibromopropionate; 1ethyl-3-[3-dimethylaminopropyl]carbodiimidehydrochloride; and succinimidyl4-[n-maleimidomethyl]-cyclohexane-1-carboxylate.

A composition disclosed herein is typically a biodegradable,bioerodible, and/or bioresorbable. In an embodiment, a silk fibroinhydrogel disclosed herein has a protein structure that makes thehydrogel resist biodegradation. In aspects of this embodiment, ahydrogel is resistant to biodegradation for, e.g., about 10 days, about20 days, about 30 days, about 40 days, about 50 days, about 60 days,about 70 days, about 80 days, or about 90 days. In other aspects of thisembodiment, a hydrogel is resistant to biodegradation for, e.g., atleast 10 days, at least 20 days, at least 30 days, at least 40 days, atleast 50 days, at least 60 days, at least 70 days, at least 80 days, orat least 90 days. In yet other aspects of this embodiment, a hydrogel isresistant to biodegradation for, e.g., about 10 days to about 30 days,about 20 days to about 50 days, about 40 days to about 60 days, about 50days to about 80 days, or about 60 days to about 90 days.

In an embodiment, a silk fibroin hydrogel disclosed herein has a proteinstructure that makes the hydrogel resist bioerosion. In aspects of thisembodiment, a hydrogel is resistant to bioerosion for, e.g., about 10days, about 20 days, about 30 days, about 40 days, about 50 days, about60 days, about 70 days, about 80 days, or about 90 days. In otheraspects of this embodiment, a hydrogel is resistant to bioerosion for,e.g., at least 10 days, at least 20 days, at least 30 days, at least 40days, at least 50 days, at least 60 days, at least 70 days, at least 80days, or at least 90 days. In yet other aspects of this embodiment, ahydrogel is resistant to bioerosion for, e.g., about 10 days to about 30days, about 20 days to about 50 days, about 40 days to about 60 days,about 50 days to about 80 days, or about 60 days to about 90 days.

In an embodiment, a silk fibroin hydrogel disclosed herein has a proteinstructure that makes the hydrogel resist bioresorption. In aspects ofthis embodiment, a hydrogel is resistant to bioresorption for, e.g.,about 10 days, about 20 days, about 30 days, about 40 days, about 50days, about 60 days, about 70 days, about 80 days, or about 90 days. Inother aspects of this embodiment, a hydrogel is resistant tobioresorption for, e.g., at least 10 days, at least 20 days, at least 30days, at least 40 days, at least 50 days, at least 60 days, at least 70days, at least 80 days, or at least 90 days. In yet other aspects ofthis embodiment, a hydrogel is resistant to bioresorption for, e.g.,about 10 days to about 30 days, about 20 days to about 50 days, about 40days to about 60 days, about 50 days to about 80 days, or about 60 daysto about 90 days.

In yet another embodiment, a silk fibroin hydrogel disclosed herein hasa protein structure that substantially includes β-turn and β-strandregions. In aspects of this embodiment, a hydrogel has a proteinstructure including, e.g., about 10% β-turn and β-strand regions, about20% β-turn and β-strand regions, about 30% β-turn and β-strand regions,about 40% β-turn and 3-strand regions, about 50% β-turn and β-strandregions, about 60% β-turn and β-strand regions, about 70% β-turn andβ-strand regions, about 80% β-turn and β-strand regions, about 90%β-turn and β-strand regions, or about 100% β-turn and β-strand regions.In other aspects of this embodiment, a hydrogel has a protein structureincluding, e.g., at least 10% β-turn and β-strand regions, at least 20%β-turn and β-strand regions, at least 30% β-turn and β-strand regions,at least 40% β-turn and β-strand regions, at least 50% β-turn andβ-strand regions, at least 60% β-turn and β-strand regions, at least 70%β-turn and β-strand regions, at least 80% β-turn and β-strand regions,at least 90% β-turn and β-strand regions, or at least 95% 3-turn andβ-strand regions. In yet other aspects of this embodiment, a hydrogelhas a protein structure including, e.g., about 10% to about 30% β-turnand β-strand regions, about 20% to about 40% β-turn and β-strandregions, about 30% to about 50% β-turn and β-strand regions, about 40%to about 60% β-turn and β-strand regions, about 50% to about 70% β-turnand β-strand regions, about 60% to about 80% β-turn and β-strandregions, about 70% to about 90% β-turn and β-strand regions, about 80%to about 100% β-turn and β-strand regions, about 10% to about 40% β-turnand β-strand regions, about 30% to about 60% β-turn and β-strandregions, about 50% to about 80% β-turn and β-strand regions, about 70%to about 100% β-turn and β-strand regions, about 40% to about 80% β-turnand β-strand regions, about 50% to about 90% β-turn and β-strandregions, about 60% to about 100% β-turn and β-strand regions, or about50% to about 100% β-turn and β-strand regions.

In yet another embodiment, a silk fibroin hydrogel disclosed herein hasa protein structure that is substantially-free of α-helix and randomcoil regions. In aspects of this embodiment, a hydrogel has a proteinstructure including, e.g., about 5% α-helix and random coil regions,about 10% α-helix and random coil regions, about 15% α-helix and randomcoil regions, about 20% α-helix and random coil regions, about 25%α-helix and random coil regions, about 30% α-helix and random coilregions, about 35% α-helix and random coil regions, about 40% α-helixand random coil regions, about 45% α-helix and random coil regions, orabout 50% α-helix and random coil regions. In other aspects of thisembodiment, a hydrogel has a protein structure including, e.g., at most5% α-helix and random coil regions, at most 10% α-helix and random coilregions, at most 15% α-helix and random coil regions, at most 20%α-helix and random coil regions, at most 25% α-helix and random coilregions, at most 30% α-helix and random coil regions, at most 35%α-helix and random coil regions, at most 40% α-helix and random coilregions, at most 45% α-helix and random coil regions, or at most 50%α-helix and random coil regions. In yet other aspects of thisembodiment, a hydrogel has a protein structure including, e.g., about 5%to about 10% α-helix and random coil regions, about 5% to about 15%α-helix and random coil regions, about 5% to about 20% α-helix andrandom coil regions, about 5% to about 25% α-helix and random coilregions, about 5% to about 30% α-helix and random coil regions, about 5%to about 40% α-helix and random coil regions, about 5% to about 50%α-helix and random coil regions, about 10% to about 20% α-helix andrandom coil regions, about 10% to about 30% α-helix and random coilregions, about 15% to about 25% α-helix and random coil regions, about15% to about 30% α-helix and random coil regions, or about 15% to about35% α-helix and random coil regions.

Aspects of the present specification provide, in part, a silk fibroinhydrogel having hardness. Hardness refers to various properties of anobject in the solid phase that gives it high resistance to various kindsof shape change when force is applied. Hardness is measured using adurometer and is a unitless value that ranges from zero to 100. Theability or inability of a hydrogel to be easily compressed will affectits suitability for application in different tissue replacement roles,i.e., mechanical compliance as bone, fat, connective tissue. Hardnesswill also affect the ability of a hydrogel to be effectively comminuted,the reason being that a hard material may be more easily andconsistently comminuted. Hardness will also affect extrudability, as asoft material may be more readily able to be slightly compressed duringinjection to pack with other particles or change shape to pass through asyringe barrel or needle.

In an embodiment, a silk fibroin hydrogel exhibits low hardness. Inaspects of this embodiment, a silk fibroin hydrogel exhibits a hardnessof, e.g., about 5, about 10, about 15, about 20, about 25, about 30, orabout 35. In other aspects of this embodiment, a silk fibroin hydrogelexhibits a hardness of, e.g., at most 5, at most 10, at most 15, at most20, at most 25, at most 30, or at most 35. In yet other aspects of thisembodiment, a silk fibroin hydrogel exhibits a hardness of, e.g., about5 to about 35, about 10 to about 35, about 15 to about 35, about 20 toabout 35, or about 25 to about 35, about 5 to about 40, about 10 toabout 40, about 15 to about 40, about 20 to about 40, about 25 to about40, or about 30 to about 40

In an embodiment, a silk fibroin hydrogel exhibits medium hardness. Inaspects of this embodiment, a silk fibroin hydrogel exhibits a hardnessof, e.g., about 40, about 45, about 50, about 55, or about 60. In otheraspects of this embodiment, a silk fibroin hydrogel exhibits a hardnessof, e.g., at least 40, at least 45, at least 50, at least 55, or atleast 60. In yet other aspects of this embodiment, a silk fibroinhydrogel exhibits a hardness of, e.g., at most 40, at most 45, at most50, at most 55, or at most 60. In still other aspects of thisembodiment, a silk fibroin hydrogel exhibits a hardness of, e.g., about35 to about 60, about 35 to about 55, about 35 to about 50, about 35 toabout 45, about 40 to about 60, about 45 to about 60, about 50 to about60, about 55 to about 60, about 40 to about 65, about 45 to about 65,about 50 to about 65, about 55 to about 65.

In another embodiment, a silk fibroin hydrogel exhibits high hardness.In aspects of this embodiment, a silk fibroin hydrogel exhibits ahardness of, e.g., about 65, about 70, about 75, about 80, about 85,about 90, about 95, or about 100. In other aspects of this embodiment, asilk fibroin hydrogel exhibits a hardness of, e.g., at least 65, atleast 70, at least 75, at least 80, at least 85, at least 90, at least95, or at least 100. In yet other aspects of this embodiment, a silkfibroin hydrogel exhibits a hardness of, e.g., about 65 to about 100,about 70 to about 100, about 75 to about 100,about 80 to about 100,about 85 to about 100, about 90 to about 100, about 65 to about 75,about 65 to about 80, about 65 to about 85, about 65 to about 90, about65 to about 95, about 60 to about 75, about 60 to about 80, about 60 toabout 85, about 60 to about 90, or about 60 to about 95.

In an embodiment, a silk fibroin hydrogel exhibits high resistant todeformation. In aspects of this embodiment, a silk fibroin hydrogelexhibits resistant to deformation of, e.g., about 100%, about 99%, about98%, about 97%, about 96%, about 95%, about 94%, about 93%, about 92%,about 91%, about 90%, about 89%, about 88%, about 87%, about 86%, orabout 85%. In other aspects of this embodiment, a silk fibroin hydrogelexhibits resistant to deformation of, e.g., at least 99%, at least 98%,at least 97%, at least 96%, at least 95%, at least 94%, at least 93%, atleast 92%, at least 91%, at least 90%, at least 89%, at least 88%, atleast 87%, at least 86%, or at least 85%. In yet other aspects of thisembodiment, a silk fibroin hydrogel exhibits resistant to deformationof, e.g., at most 99%, at most 98%, at most 97%, at most 96%, at most95%, at most 94%, at most 93%, at most 92%, at most 91%, at most 90%, atmost 89%, at most 88%, at most 87%, at most 86%, or at most 85%. Instill aspects of this embodiment, a silk fibroin hydrogel exhibitsresistant to deformation of, e.g., about 85% to about 100%, about 87% toabout 100%, about 90% to about 100%, about 93% to about 100%, about 95%to about 100%, or about 97% to about 100%.

A silk fibroin hydrogel exhibits an elastic modulus. Elastic modulus, ormodulus of elasticity, refers to the ability of a hydrogel material toresists deformation, or, conversely, an object's tendency to benon-permanently deformed when a force is applied to it. The elasticmodulus of an object is defined as the slope of its stress-strain curvein the elastic deformation region: λ=stress/strain, where λ is theelastic modulus in Pascal's; stress is the force causing the deformationdivided by the area to which the force is applied; and strain is theratio of the change caused by the stress to the original state of theobject. Specifying how stresses are to be measured, includingdirections, allows for many types of elastic moduli to be defined. Thethree primary elastic moduli are tensile modulus, shear modulus, andbulk modulus.

Tensile modulus (E) or Young's modulus is an objects response to linearstrain, or the tendency of an object to deform along an axis whenopposing forces are applied along that axis. It is defined as the ratioof tensile stress to tensile strain. it is often referred to simply asthe elastic modulus. The shear modulus or modulus of rigidity refers toan object's tendency to shear (the deformation of shape at constantvolume) when acted upon by opposing forces. It is defined as shearstress over shear strain. The shear modulus is part of the derivation ofviscosity. The shear modulus is concerned with the deformation of asolid when it experiences a force parallel to one of its surfaces whileits opposite face experiences an opposing force (such as friction). Thebulk modulus (K) describes volumetric elasticity or an object'sresistance to uniform compression, and is the tendency of an object todeform in all directions when uniformly loaded in all directions. it isdefined as volumetric stress over volumetric strain, and is the inverseof compressibility. The bulk modulus is an extension of Young's modulusto three dimensions.

In another embodiment, a silk fibroin hydrogel exhibits a tensilemodulus. in aspects of this embodiment, a silk fibroin hydrogel exhibitsa tensile modulus of, e.g., about 1 MPa, about 10 MPa, about 20 MPa,about 30 MPa, about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa,about 80 MPa, about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa,about 400 MPa, about 500 MPa, about 750 MPa, about 1 GPa, about 5 GPa,about 10 GPa, about 15 GPa, about 20 GPa, about 25 GPa,or about 30 GPa.In other aspects of this embodiment, a silk fibroin hydrogel exhibits atensile modulus of, e.g., at least 1 MPa, at least 10 MPa, at least 20MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa,at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, atleast 200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa, atleast 750 MPa, at least 1 GPa, at least 5 GPa, at least 10 GPa, at least15 GPa, at least 20 GPa, at least 25 GPa,or at least 30 GPa In yet otheraspects of this embodiment, a silk fibroin hydrogel exhibits a tensilemodulus of, e.g., about 1 MPa to about 30 MPa, about 10 MPa to about 50MPa, about 25 MPa to about 75 MPa, about 50 MPa to about 100 MPa, about100 MPa to about 300 MPa, about 200 MPa to about 400 MPa, about 300 MPato about 500 MPa, about 100 MPa to about 500 MPa, about 250 MPa to about750 MPa, about 500 MPa to about 1 GPa, about 1GPa to about 30 GPa, about10 GPa to about 30 GPa.

In another embodiment, a silk fibroin hydrogel exhibits shear modulus.In aspects of this embodiment, a silk fibroin hydrogel exhibits a shearmodulus of, e.g., about 1 MPa, about 10 MPa, about 20 MPa, about 30 MPa,about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa,about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about 400MPa, about 500 MPa, about 750 MPa, about 1 GPa, about 5 GPa, about 10GPa, about 15 GPa, about 20 GPa, about 25 GPa, or about 30 GPa. In otheraspects of this embodiment, a silk fibroin hydrogel exhibits a shearmodulus of, e.g., at least 1 MPa, at least 10 MPa, at least 20 MPa, atleast 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa, atleast 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, atleast 200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa, atleast 750 MPa, at least 1 GPa, at least 5 GPa, at least 10 GPa, at least15 GPa, at least 20 GPa, at least 25 GPa,or at least 30 GPa In yet otheraspects of this embodiment, a silk fibroin hydrogel exhibits a shearmodulus of, e.g., about 1 MPa to about 30 MPa, about 10 MPa to about 50MPa, about 25 MPa to about 75 MPa, about 50 MPa to about 100 MPa, about100 MPa to about 300 MPa, about 200 MPa to about 400 MPa, about 300 MPato about 500 MPa, about 100 MPa to about 500 MPa, about 250 MPa to about750 MPa, about 500 MPa to about 1 GPa, about 1GPa to about 30 GPa, about10 GPa to about 30 GPa.

In another embodiment, a silk fibroin hydrogel exhibits a bulk modulus.In aspects of this embodiment, a silk fibroin hydrogel exhibits a bulkmodulus of, e.g., about 5 GPa, about 6 GPa, about 7 GPa, about 8 GPa,about 9 GPa, about 10 GPa, about 15 GPa, about 20 GPa, about 25 GPa,about 30 GPa, about 35 GPa, about 40 GPa, about 45 GPa, about 50 GPa,about 60 GPa, about 70 GPa, about 80 GPa, about 90 GPa, about 100 GPa.In other aspects of this embodiment, a silk fibroin hydrogel exhibits abulk modulus of, e.g., at least 5 GPa, at least 6 GPa, at least 7 GPa,at least 8 GPa, at least 9 GPa, at least 10 GPa, at least 15 GPa, atleast 20 GPa, at least 25 GPa, at least 30 GPa, at least 35 GPa, atleast 40 GPa, at least 45 GPa, at least 50 GPa, at least 60 GPa, atleast 70 GPa, at least 80 GPa, at least 90 GPa, at least 100 GPa. In yetother aspects of this embodiment, a silk fibroin hydrogel exhibits abulk modulus of, e.g., about 5 GPa to about 50 GPa, about 5 GPa to about100 GPa, about 10 GPa to about 50 GPa, about 10 GPa to about 100 GPa, orabout 50 GPa to about 100 GPa.

A silk fibroin hydrogel exhibits high tensile strength. Tensile strengthhas three different definitional points of stress maxima. Yield strengthrefers to the stress at which material strain changes from elasticdeformation to plastic deformation, causing it to deform permanently.Ultimate strength refers to the maximum stress a material can withstandwhen subjected to tension, compression or shearing. It is the maximumstress on the stress-strain curve. Breaking strength refers to thestress coordinate on the stress-strain curve at the point of rupture, orwhen the material pulls apart.

In another embodiment, a silk fibroin hydrogel exhibits high yieldstrength relative to other polymer classes. In aspects of thisembodiment, a silk fibroin hydrogel exhibits a yield strength of, e.g.,about 0.1 MPa, about 0.5 MPa, about 1 MPa, about 5 MPa, about 10 MPa,about 20 MPa, about 30 MPa, about 40 MPa, about 50 MPa, about 60 MPa,about 70 MPa, about 80 MPa, about 90 MPa, about 100 MPa, about 200 MPa,about 300 MPa, about 400 MPa, about 500 MPa. In other aspects of thisembodiment, a silk fibroin hydrogel exhibits a yield strength of, e.g.,at least 0.1 MPa, at least 0.5 MPa, at least 1 MPa, at least 5 MPa, atleast 10 MPa, at least 20 MPa, at least 30 MPa, at least 40 MPa, atleast 50 MPa, at least 60 MPa, at least 70 MPa, at least 80 MPa, atleast 90 MPa, at least 100 MPa, at least 200 MPa, at least 300 MPa, atleast 400 MPa, at least 500 MPa. In yet other aspects of thisembodiment, a silk fibroin hydrogel exhibits a yield strength of, e.g.,at most 1 MPa, at most 5 MPa, at most 10 MPa, at most 20 MPa, at most 30MPa, at most 40 MPa, at most 50 MPa, at most 60 MPa, at most 70 MPa, atmost 80 MPa, at most 90 MPa, at most 100 MPa, at most 200 MPa, at most300 MPa, at most 400 MPa, at most 500 MPa, at most 600 MPa, at most 700MPa, at most 800 MPa, at most 900 MPa, at most 1000 MPa, at most 1500MPa, or at most 2000 MPa. In still other aspects of this embodiment, asilk fibroin hydrogel exhibits a yield strength of, e.g., about 1 MPa toabout 50 MPa, about 1 MPa to about 60 MPa, about 1 MPa to about 70 MPa,about 1 MPa to about 80 MPa, about 1 MPa to about 90 MPa, about 1 MPa toabout 100 MPa, about 10 MPa to about 50 MPa, about 10 MPa to about 60MPa, about 10 MPa to about 70 MPa, about 10 MPa to about 80 MPa, about10 MPa to about 90 MPa, about 10 MPa to about 100 MPa, about 10 MPa toabout 200 MPa, about 10 MPa to about 300 MPa, or about 100 MPa to about300 MPa.

In another embodiment, a silk fibroin hydrogel exhibits high ultimatestrength. In aspects of this embodiment, a silk fibroin hydrogelexhibits an ultimate strength of, e.g., about 0.1 MPa, about 0.5 MPa,about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 30 MPa,about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa,about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about 400MPa, about 500 MPa. In other aspects of this embodiment, a silk fibroinhydrogel exhibits an ultimate strength of, e.g., at least 0.1 MPa, atleast 0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, at least20 MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60MPa, at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100MPa, at least 200 MPa, at least 300 MPa, at least 400 MPa, at least 500MPa. In yet other aspects of this embodiment, a silk fibroin hydrogelexhibits an ultimate strength of, e.g., at most 1 MPa, at most 5 MPa, atmost 10 MPa, at most 20 MPa, at most 30 MPa, at most 40 MPa, at most 50MPa, at most 60 MPa, at most 70 MPa, at most 80 MPa, at most 90 MPa, atmost 100 MPa, at most 200 MPa, at most 300 MPa, at most 400 MPa, at most500 MPa, at most 600 MPa, at most 700 MPa, at most 800 MPa, at most 900MPa, at most 1000 MPa, at most 1500 MPa, or at most 2000 MPa. In stillother aspects of this embodiment, a silk fibroin hydrogel exhibits anultimate strength of, e.g., about 1 MPa to about 50 MPa, about 1 MPa toabout 60 MPa, about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa,about 1 MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPato about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about 70MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa, about10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about 10 MPa toabout 300 MPa, or about 100 MPa to about 300 MPa.

In another embodiment, a silk fibroin hydrogel exhibits high breakingstrength. In aspects of this embodiment, a silk fibroin hydrogelexhibits a breaking strength of, e.g., about 0.1 MPa, about 0.5 MPa,about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 30 MPa,about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa,about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about 400MPa, about 500 MPa. In other aspects of this embodiment, a silk fibroinhydrogel exhibits a breaking strength of, e.g., at least 0.1 MPa, atleast 0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, at least20 MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60MPa, at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100MPa, at least 200 MPa, at least 300 MPa, at least 400 MPa, at least 500MPa. In yet other aspects of this embodiment, a silk fibroin hydrogelexhibits a breaking strength of, e.g., at most 1 MPa, at most 5 MPa, atmost 10 MPa, at most 20 MPa, at most 30 MPa, at most 40 MPa, at most 50MPa, at most 60 MPa, at most 70 MPa, at most 80 MPa, at most 90 MPa, atmost 100 MPa, at most 200 MPa, at most 300 MPa, at most 400 MPa, at most500 MPa, at most 600 MPa, at most 700 MPa, at most 800 MPa, at most 900MPa, at most 1000 MPa, at most 1500 MPa, or at most 2000 MPa. In stillother aspects of this embodiment, a silk fibroin hydrogel exhibits abreaking strength of, e.g., about 1 MPa to about 50 MPa, about 1 MPa toabout 60 MPa, about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa,about 1 MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPato about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about 70MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa, about10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about 10 MPa toabout 300 MPa, or about 100 MPa to about 300 MPa.

Aspects of the present specification provide, in part, a silk fibroinhydrogel having a transparency and/or translucency. Transparency (alsocalled pellucidity or diaphaneity) is the physical property of allowinglight to pass through a material, whereas translucency (also calledtranslucence or translucidity) only allows light to pass throughdiffusely. The opposite property is opacity. Transparent materials areclear, while translucent ones cannot be seen through clearly. The silkfibroin hydrogels disclosed herein may, or may not, exhibit opticalproperties such as transparency and translucency. In certain cases,e.g., superficial line filling, it would be an advantage to have anopaque hydrogel. In other cases such as development of a lens or a“humor” for filling the eye, it would be an advantage to have atranslucent hydrogel. These properties could be modified by affectingthe structural distribution of the hydrogel material. Factors used tocontrol a hydrogel's optical properties include, without limitation,silk fibroin concentration, gel crystallinity, and hydrogel homogeneity.

When light encounters a material, it can interact with it in severaldifferent ways. These interactions depend on the nature of the light(its wavelength, frequency, energy, etc.) and the nature of thematerial. Light waves interact with an object by some combination ofreflection, and transmittance with refraction. As such, an opticallytransparent material allows much of the light that falls on it to betransmitted, with little light being reflected. Materials which do notallow the transmission of light are called optically opaque or simplyopaque.

In an embodiment, a silk fibroin hydrogel is optically transparent. Inaspects of this embodiment, a silk fibroin hydrogel transmits, e.g.,about 75% of the light, about 80% of the light, about 85% of the light,about 90% of the light, about 95% of the light, or about 100% of thelight. In other aspects of this embodiment, a silk fibroin hydrogeltransmits, e.g., at least 75% of the light, at least 80% of the light,at least 85% of the light, at least 90% of the light, or at least 95% ofthe light. In yet other aspects of this embodiment, a silk fibroinhydrogel transmits, e.g., about 75% to about 100% of the light, about80% to about 100% of the light, about 85% to about 100% of the light,about 90% to about 100% of the light, or about 95% to about 100% of thelight.

In another embodiment, a silk fibroin hydrogel is optically opaque. Inaspects of this embodiment, a silk fibroin hydrogel transmits, e.g.,about 5% of the light, about 10% of the light, about 15% of the light,about 20% of the light, about 25% of the light, about 30% of the light,about 35% of the light, about 40% of the light, about 45% of the light,about 50% of the light, about 55% of the light, about 60% of the light,about 65% of the light, or about 70% of the light. In other aspects ofthis embodiment, a silk fibroin hydrogel transmits, e.g., at most 5% ofthe light, at most 10% of the light, at most 15% of the light, at most20% of the light, at most 25% of the light, at most 30% of the light, atmost 35% of the light, at most 40% of the light, at most 45% of thelight, at most 50% of the light, at most 55% of the light, at most 60%of the light, at most 65% of the light, at most 70% of the light, or atmost 75% of the light. In other aspects of this embodiment, a silkfibroin hydrogel transmits, e.g., about 5% to about 15%, about 5% toabout 20%, about 5% to about 25%, about 5% to about 30%, about 5% toabout 35%, about 5% to about 40%, about 5% to about 45%, about 5% toabout 50%, about 5% to about 55%, about 5% to about 60%, about 5% toabout 65%, about 5% to about 70%, about 5% to about 75%, about 15% toabout 20%, about 15% to about 25%, about 15% to about 30%, about 15% toabout 35%, about 15% to about 40%, about 15% to about 45%, about 15% toabout 50%, about 15% to about 55%, about 15% to about 60%, about 15% toabout 65%, about 15% to about 70%, about 15% to about 75%, about 25% toabout 35%, about 25% to about 40%, about 25% to about 45%, about 25% toabout 50%, about 25% to about 55%, about 25% to about 60%, about 25% toabout 65%, about 25% to about 70%, or about 25% to about 75%, of thelight.

In an embodiment, a silk fibroin hydrogel is optically translucent. Inaspects of this embodiment, a silk fibroin hydrogel diffusely transmits,e.g., about 75% of the light, about 80% of the light, about 85% of thelight, about 90% of the light, about 95% of the light, or about 100% ofthe light. In other aspects of this embodiment, a silk fibroin hydrogeldiffusely transmits, e.g., at least 75% of the light, at least 80% ofthe light, at least 85% of the light, at least 90% of the light, or atleast 95% of the light. In yet other aspects of this embodiment, a silkfibroin hydrogel diffusely transmits, e.g., about 75% to about 100% ofthe light, about 80% to about 100% of the light, about 85% to about 100%of the light, about 90% to about 100% of the light, or about 95% toabout 100% of the light.

After formation of a hydrogel described herein, the hydrogel can furtherprocessed. For example, to remove enhancer species and become a morecomplete, the formed hydrogel may be leeched against a solvent, such as,e.g., water, under ambient temperature and pressure conditions for threedays with five changes of water. The hydrogel may be leeched againstultra-pure water of a volume at least 100-times that of the gel. Morespecifically, for example, the gels may be placed in a bulk of purifiedwater and the rinse changed at hours 12, 24 and 48 with 15 mL gel per1.5 L water. The number of rinses and volume ratios involved may bealtered so long as the resultant hydrogel is substantially free ofresidual gelation enhancer.

A hydrogel may be further processed by pulverizing the hydrogel intoparticles and mixed with a carrier phase such as, e.g., water or asaline solution to form an injectable or topical substance like asolution, oil, lotion, gel, ointment, cream, slurry, salve, or paste. Ahydrogel may be milled to a particle size from about 10 μm to about 1000μm in diameter, such as 15 μm to 30 μm. Saline is then added as acarrier phase by first determining the bulk volume of a hydrogel, thenvigorously pulverizing the hydrogel into particles while incorporatingan appropriate volume of saline to achieve a desired carrier to hydrogelparticle ratio. For example, hydrogel milling may be accomplished bymeans of a forced sieving of bulk hydrogel through a series of stainlesssteel cloth sieves of decreasing pore sizes. In another example, ahydrogel may be loaded into a syringe and pulverized with a spatula to afine paste with saline.

A composition disclosed herein may be formulated using materialprocessing constraints such as silk concentration and salineconcentration to tailor material longevity in vivo. In one example, asilk hydrogel might be tailored for a persistence of five weeks to sixweeks in vivo by using a 1%-3% (w/v) silk gel with 25%-50% (v/v) salinecarrier. In another example, a silk hydrogel might be tailored for apersistence of two months to three months in vivo by using a 3%-5% (w/v)silk gel with 20%-40% (v/v) saline. In another example, a silk hydrogelmight be tailored for a persistence of 5-6 months by using 4-6% (w/v)silk gel with 20-40% (v/v) saline. In another example, a silk hydrogelmight be tailored for a persistence of 7-10 months by using a 6-8% (w/v)silk gel with 20-30% (v/v) saline. The persistence of these materialsmight also be increased or decreased by increasing or decreasingparticle size respectively.

Gel emulsion saline content and gel silk concentration could be used tomodify the mechanical profile of the silk gel materials for particularapplications. For example, a gel emulsion of about 1% (w/v) to about 5%(w/v) silk gel concentration with 5%-95% lubricant (e.g., 5%-95% (w/v)saline/PBS) may be useful as a dermal filler, bulking agent, camouflageagent, intramuscular or sub-Q filler, or pharmaceutical delivery vector.A gel emulsion of, for example, about 5% (w/v) to about 8% (w/v) silkgel concentration with 0% to about 30% lubricant fluid may be useful inbone defects or cartilage defects.

Aspects of the present specification provide, in part, a compositioncomprising a gel phase including a hydrogel comprising a matrix polymer.The compositions disclosed herein can further comprise a hydrogelcomprising one or more matrix polymers in addition to hydrogel particlescomprising silk fibroin, or a hydrogel comprising one or more matrixpolymers and silk fibroin. As used herein, the term “matrix polymer”refers to a polymer that can become part of and/or function as anextracellular matrix polymer and pharmaceutically acceptable saltsthereof. Non-limiting examples of a matrix polymer include aglycosaminoglycan like chondroitin sulfate, dermatan sulfate, keratansulfate, hyaluronan; a lubricin; a polysaccharide, and an elasticprotein (like silk protein, resilin, resilin-like polypeptides (RLPs),elastin (including tropoelastin, fibrillin and fibullin), elastin-likepolypeptides (ELPs), gluten (including gliadin and glutenin), abductin,byssus, and collagen). Non-limiting examples of a pharmaceuticallyacceptable salt of a matrix polymer includes sodium salts, potassiumsalts, magnesium salts, calcium salts, and combinations thereof. Matrixpolymers useful in the compositions and methods disclosed herein aredescribed in, e.g., Piron and Tholin, Polysaccharide Crosslinking,Hydrogel Preparation, Resulting Polysaccharides(s) and Hydrogel(s), usesThereof, U.S. Patent Publication 2003/0148995; Lebreton, Cross-Linkingof Low and High Molecular Weight Polysaccharides Preparation ofInjectable Monophase Hydrogels; Lebreton, Viscoelastic SolutionsContaining Sodium Hyaluronate and Hydroxypropyl Methyl Cellulose,Preparation and Uses, U.S. Patent Publication 2008/0089918; Lebreton,Hyaluronic Acid-Based Gels Including Lidocaine, U.S. Patent Publication2010/0028438; and Polysaccharides and Hydrogels thus Obtained, U.S.Patent Publication 2006/0194758; and Di Napoli, Composition and Methodfor Intradermal Soft Tissue Augmentation, International PatentPublication WO 2004/073759, each of which is hereby incorporated byreference in its entirety.

Aspects of the present specification provide, in part, a compositioncomprising a glycosaminoglycan. As used herein, the term“glycosaminoglycan” is synonymous with “GAG” and “mucopolysaccharide”and refers to long unbranched polysaccharides consisting of a repeatingdisaccharide units. The repeating unit consists of a hexose (six-carbonsugar) or a hexuronic acid, linked to a hexosamine (six-carbon sugarcontaining nitrogen) and pharmaceutically acceptable salts thereof.Members of the GAG family vary in the type of hexosamine, hexose orhexuronic acid unit they contain, such as, e.g., glucuronic acid,iduronic acid, galactose, galactosamine, glucosamine) and may also varyin the geometry of the glycosidic linkage. Any glycosaminoglycan isuseful in the compositions disclosed herein with the proviso that theglycosaminoglycan improves a condition of the skin, such as, e.g.,hydration or elasticity. GAGs useful in the compositions and methodsdisclosed herein are commercially available. Table 1 listsrepresentative GAGs.

TABLE 1 Examples of GAGs Glycosidic Hexuronic linkage Name acid/HexoseHexosamine geometry Unique features Chondroitin GlcUA or GalNAc or-4GlcUAβ1- Most prevalent GAG sulfate GlcUA(2S) GalNAc(4S) or 3GalNAcβ1-GalNAc(6S) or GalNAc(4S,6S) Dermatan GlcUA or GalNAc or -4IdoUAβ1-Distinguished from chondroitin sulfate IdoUA or GalNAc(4S) or 3GalNAcβ1-sulfate by the presence of IdoUA(2S) GalNAc(6S) or iduronic acid,although some GalNAc(4S,6S) hexuronic acid monosaccharides may beglucuronic acid. Keratan Gal or GlcNAc or -3Gal(6S)β1- Keratan sulfatetype II may be sulfate Gal(6S) GlcNAc(6S) 4GlcNAc(6S)β1- fucosylated.Heparin GlcUA or GlcNAc or -4IdoUA(2S)α1- Highest negative chargeIdoUA(2S) GlcNS or 4GlcNS(6S)α1- density of any known GlcNAc(6S) orbiological molecule GlcNS(6S) Heparan GlcUA or GlcNAc or -4GlcUAβ1-Highly similar in structure to sulfate IdoUA or GlcNS or 4GlcNAcα1-heparin, however heparan IdoUA(2S) GlcNAc(6S) or sulfates disaccharideunits are GlcNS(6S) organised into distinct sulfated and non-sulfateddomains. Hyaluronan GlcUA GlcNAc -4GlcUAβ1- The only GAG that is3GlcNAcβ1- exclusively non-sulfated GlcUA = β-D-glucuronic acidGlcUA(2S) = 2-O-sulfo-β-D-glucuronic acid IdoUA = α-L-iduronic acidIdoUA(2S) = 2-O-sulfo-α-L-iduronic acid Gal = β-D-galactose Gal(6S) =6-O-sulfo-β-D-galactose GalNAc = β-D-N-acetylgalactosamine GalNAc(4S) =β-D-N-acetylgalactosamine-4-O-sulfate GalNAc(6S) =β-D-N-acetylgalactosamine-6-O-sulfate GalNAc(4S,6S) =β-D-N-acetylgalactosamine-4-O, 6-O-sulfate GlcNAc =α-D-N-acetylglucosamine GlcNS = α-D-N-sulfoglucosamine GlcNS(6S) =α-D-N-sulfoglucosamine-6-O-sulfate

Aspects of the present specification provide, in part, a compositioncomprising a chondroitin sulfate. As used herein, the term “chondroitinsulfate” refers to an unbranched, sulfated GAG of variable lengthcomprising disaccharides of two alternating monosaccharides ofD-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc) andpharmaceutically acceptable salts thereof. A chondroitin sulfate mayalso include D-glucuronic acid residues that are epimerized intoL-iduronic acid (IdoA), in which case the resulting disaccharide isreferred to as dermatan sulfate. A chondroitin sulfate polymer can havea chain of over 100 individual sugars, each of which can be sulfated invariable positions and quantities. Chondroitin sulfate is an importantstructural component of cartilage and provides much of its resistance tocompression. Any chondroitin sulfate is useful in the compositionsdisclosed herein with the proviso that the chondroitin sulfate improvesa condition of the skin, such as, e.g., hydration or elasticity.Non-limiting examples of pharmaceutically acceptable salts ofchondroitin sulfate include sodium chondroitin sulfate, potassiumchondroitin sulfate, magnesium chondroitin sulfate, calcium chondroitinsulfate, and combinations thereof.

Aspects of the present specification provide, in part, a compositioncomprising a keratan sulfate. As used herein, the term “keratan sulfate”refers to a GAG of variable length comprising disaccharide units, whichthemselves include β-D-galactose and N-acetyl-D-gafactosamine (GaINAc)and pharmaceutically acceptable salts thereof. Disaccharides within therepeating region of keratan sulfate may be fucosylated andN-Acetylneuraminic acid caps the end of the chains. Any keratan sulfateis useful in the compositions disclosed herein with the proviso that thekeratan sulfate improves a condition of the skin, such as, e.g.,hydration or elasticity. Non-limiting examples of pharmaceuticallyacceptable salts of keratan sulfate include sodium keratan sulfate,potassium keratan sulfate, magnesium keratan sulfate, calcium keratansulfate, and combinations thereof.

Aspects of the present specification provide, in part, a compositioncomprising a hyaluronan. As used herein, the term “hyaluronic acid” issynonymous with “HA”, “hyaluronic acid”, and “hyaluronate” refers to ananionic, non-sulfated glycosaminoglycan polymer comprising disaccharideunits, which themselves include D-glucuronic acid andD-N-acetylglucosamine monomers, linked together via alternating β-1,4and β-1,3 glycosidic bonds and pharmaceutically acceptable saltsthereof. Hyaluronan can be purified from animal and non-animal sources.Polymers of hyaluronan can range in size from about 5,000 Da to about20,000,000 Da. Any hyaluronan is useful in the compositions disclosedherein with the proviso that the hyaluronan improves a condition of theskin, such as, e.g., hydration or elasticity. Non-limiting examples ofpharmaceutically acceptable salts of hyaluronan include sodiumhyaluronan, potassium hyaluronan, magnesium hyaluronan, calciumhyaluronan, and combinations thereof.

Aspects of the present specification provide, in part, a compositioncomprising a lubricin. As used herein, the term “lubricin” is synonymouswith “proteoglycan 4” or superficial zone protein” and refers to alarge, water soluble glycoprotein encoded by the PRG4 gene andpharmaceutically acceptable salts thereof. Lubricin is about 345 kDaapproximately equal proportions of protein and the GAGs chondroitinsulfate and keratan sulfate. The structure of lubricin molecule is thatof a partially extended flexible rod and, in solution, occupies asmaller spatial domain than would be expected from structuralpredictions. This characteristic may aid in the molecule's boundarylubricating ability. Lubricin is present in synovial fluid and on thesurface (superficial layer) of articular cartilage and therefore playsan important role in joint lubrication and synovial homeostasis. Theexpression of lubricin has also been detected and the protein localizedin tendon, meniscus, lung, liver, heart, bone, ligament, muscle andskin. Any lubricin is useful in the compositions disclosed herein withthe proviso that the lubricin improves a condition of the skin, such as,e.g., hydration or elasticity. Non-limiting examples of pharmaceuticallyacceptable salts of lubricin include sodium lubricin, potassiumlubricin, magnesium lubricin, calcium lubricin, and combinationsthereof.

Aspects of the present pharmaceutical compositions provide, in part, apolysaccharide. As used herein, the term “polysaccharide” refers to highmolecular weight compounds comprising at least eleven monosaccharideunits. Polysaccharides consisting of only one kind of repeating unit arecalled homo polysaccharide polymers, whereas polysaccharides formed fromtwo or more different repeating units and called co polysaccharidepolymers. A polysaccharide can be natural or synthetic. Non-limitingexamples of polysaccharide include, e.g., dextrans (like dextran 1K,dextran 4K, dextran 40K, dextran 60K, and dextran 70K), dextrin,glycogen, inulin, starch, starch derivatives (like hydroxymethyl starch,hydroxyethyl starch, hydroxypropyl starch, hydroxybutyl starch, andhydroxypentyl starch), hetastarch, cellulose, MOLL, methyl cellulose(MC), carboxymethyl cellulose (CMC), hydroxyethyl cellulose (HEC),hydroxypropyl cellulose (HPC), hydroxyethyl methyl cellulose (HEMC),hydroxypropyl methyl cellulose (HPMC); polyvinyl acetates (PVA);polyvinyl pyrrolidones (PVP), also known as povidones, having a K-valueof less than or equal to 18, a K-value greater than 18 or less than orequal to 95, or a K-value greater than 95, like PVP 12 (KOLLIDON® 12),PVP 17 (KOLLIDON® 17), PVP 25 (KOLLIDON® 25), PVP 30 (KOLLIDON® 30), PVP90 (KOLLIDON® 90); polyethylene glycols like PEG 100, PEG 200, PEG 300,PEG 400, PEG 500, PEG 600, PEG 700, PEG, 800, PEG 900, PEG 1000, PEG1100, PEG 1200, PEG 1300, PEG 1400, PEG 1500, PEG 1600, PEG 1700, PEG1800, PEG 1900, PEG 2000, PEG 2100, PEG 2200, PEG 2300, PEG 2400, PEG2500, PEG 2600, PEG 2700, PEG 2800, PEG 2900, PEG 3000, PEG 3250, PEG3350, PEG 3500, PEG 3750, PEG 4000, PEG 4250, PEG 4500, PEG 4750, PEG5000, PEG 5500, PEG 6000, PEG 6500, PEG 75000, PEG 7500, or PEG 8000;and polyethylene imines (PEI).

Aspects of the present specification provide, in part, a compositioncomprising an elastic protein. As used herein, the term “elasticprotein” refers to a structural polypeptide with a wide range ofphysical properties including, without limitation, high resilience inthat the polypeptide can be deformed reversibly without loss of energy,the ability to be deformed to large strains with little force, and/orwith low stiffness in that the polypeptide can be stretched. Forexample, elastic proteins like elastin and resilin have a combination ofhigh resilience, large strains and low stiffness is characteristic ofrubber-like proteins that function in the storage of elastic-strainenergy. Other elastic proteins, like collagen, provides exceptionalenergy storage capacity but are not very stretchy. Mussel byssus threadsand spider dragline silks are also elastic proteins because they areremarkably stretchy, in spite of their considerable strength, lowresilience, and stiffness. The silk fibroin disclosed herein is anotherelastic protein. Non-limiting examples of elastic proteins include silkprotein, resilin, resilin-like polypeptides (RLPs), elastin (includingtropoelastin, fibrillin and fibullin), elastin-like polypeptides (ELPs),gluten (including gliadin and glutenin), abductin, byssus, and collagen.In general, elastic proteins have at least one domain containing elasticrepeat motifs and another non-elastic domain where crosslinks can beformed. See, e.g., Tatham and Shewry, Comparative Structures andProperties of Elastic Proteins, Phil. Trans. R. Soc. Lond. B 357:229-234 (2002), which is hereby incorporated by reference in itsentirety. However, both resilin and abductin are exceptions sincecrosslinking can occur within the elastic repeat motif.

Aspects of the present specification provide, in part, a compositioncomprising a crosslinked matrix polymer. As used herein, the term“crosslinked” refers to the intermolecular bonds joining the individualpolymer molecules, or monomer chains, into a more stable structure likea gel. As such, a crosslinked matrix polymer has at least oneintermolecular bond joining at least one individual polymer molecule toanother one. Matrix polymers disclosed herein may be crosslinked usingdialdehydes and disuf ides crosslinking agents including, withoutlimitation, multifunctional PEG-based crosslinking agents, divinylsulfones, diglycidyl ethers, and bis-epoxides. Non-limiting examples ofhyaluronan crosslinking agents include divinyl sulfone (DVS),1,4-butanediol diglycidyl ether (BDDE),1,2-bis(2,3-epoxypropoxy)ethylene (EG DG E), 1,2,7,8-diepoxyoctane(DEO), biscarbodiimide (BCDI), pentaerythritol tetraglycidyl ether(PETGE), adipic dihydrazide (ADH), bis(sulfosuccinimidyl)suberate (BS),hexamethylenediamine (HMDA), 1-(2,3-epoxypropyl)-2,3-epoxycyclohexane,or combinations thereof.

Aspects of the present specification provide, in part, a compositioncomprising a crosslinked matrix polymer having a degree of crosslinking.As used herein, the term “degree of crosslinking” refers to thepercentage of matrix polymer monomeric units that are bound to across-linking agent, such as, e.g., the disaccharide monomer units ofhyaluronan. Thus, a composition that that has a crosslinked matrixpolymer with a 4% degree of crosslinking means that on average there arefour crosslinking molecules for every 100 monomeric units. Every otherparameter being equal, the greater the degree of crosslinking, theharder the gel becomes. Non-limiting examples of a degree ofcrosslinking include about 1% to about 15%.

In an embodiment, a composition comprises a crosslinked matrix polymer.In other aspects of this embodiment, a composition comprises acrosslinked matrix polymer where the partially crosslinked matrixpolymer represents, e.g., about 1% by weight, about 2% by weight, about3% by weight, about 4% by weight, about 5% by weight, about 6% byweight, about 7% by weight, about 8% by weight, or about 9%, or about10% by weight, of the total matrix polymer present in the composition.In yet other aspects of this embodiment, a composition comprises acrosslinked matrix polymer where the partially crosslinked matrixpolymer represents, e.g., at most 1% by weight, at most 2% by weight, atmost 3% by weight, at most 4% by weight, at most 5% by weight, at most6% by weight, at most 7% by weight, at most 8% by weight, at most 9% byweight, or at most 10% by weight, of the total matrix polymer present inthe composition. In still other aspects of this embodiment, acomposition comprises a crosslinked matrix polymer where the partiallycrosslinked matrix polymer represents, e.g., about 0% to about 10% byweight, about 1% to about 10% by weight, about 3% to about 10% byweight, or about 5% to about 10% by weight, of the total matrix polymerpresent in the composition.

In other aspects of this embodiment, a composition comprises acrosslinked matrix polymer where the degree of crosslinking is about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, orabout 15%. In yet other aspects of this embodiment, a compositioncomprises a crosslinked matrix polymer where the degree of crosslinkingis at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, atmost 12%, at most 13%, at most 14%, or at most 15%. In still otheraspects of this embodiment, a composition comprises a crosslinked matrixpolymer where the degree of crosslinking is about 1% to about 15%, about2% to about 11%, about 3% to about 10%, about 1% to about 5%, about 10%to about 15%, about 11% to about 15%, about 6% to about 10%, or about 6%to about 8%.

In another embodiment, a composition comprises a crosslinkedglycosaminoglycan. In aspect of this embodiment, a composition comprisesa crosslinked chondroitin sulfate polymer, a crosslinked dermatansulfate polymer, a crosslinked keratan sulfate polymer, a crosslinkedheparan polymer, a crosslinked heparan sulfate polymer, or a crosslinkedhyaluronan polymer. In other aspects of this embodiment, a compositioncomprises a crosslinked glycosaminoglycan where the crosslinkedglycosaminoglycan represents, e.g., about 1% by weight, about 2% byweight, about 3% by weight, about 4% by weight, about 5% by weight,about 6% by weight, about 7% by weight, about 8% by weight, or about 9%,or about 10% by weight, of the total glycosaminoglycan present in thecomposition. In yet other aspects of this embodiment, a fluidcomposition comprises a crosslinked glycosaminoglycan where thecrosslinked glycosaminoglycan represents, e.g., at most 1% by weight, atmost 2% by weight, at most 3% by weight, at most 4% by weight, at most5% by weight, at most 6% by weight, at most 7% by weight, at most 8% byweight, at most 9% by weight, or at most 10% by weight, of the totalglycosaminoglycan present in the composition. In still other aspects ofthis embodiment, a composition comprises a crosslinked glycosaminoglycanwhere the crosslinked glycosaminoglycan represents, e.g., about 0% toabout 10% by weight, about 1% to about 10% by weight, about 3% to about10% by weight, or about 5% to about 10% by weight, of the totalglycosaminoglycan present in the composition.

In other aspects of this embodiment, a composition comprises acrosslinked glycosaminoglycan where the degree of crosslinking is about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, orabout 15%. In yet other aspects of this embodiment, a compositioncomprises a crosslinked glycosaminoglycan where the degree ofcrosslinking is at most 1%, at most 2%, at most 3%, at most 4%, at most5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most11%, at most 12%, at most 13%, at most 14%, or at most 15%. In stillother aspects of this embodiment, a composition comprises a crosslinkedglycosaminoglycan where the degree of crosslinking is about 1% to about15%, about 2% to about 11%, about 3% to about 10%, about 1% to about 5%,about 10% to about 15%, about 11% to about 15%, about 6% to about 10%,or about 6% to about 8%.

In yet another embodiment, a composition comprises a crosslinkedlubricin. In aspects of this embodiment, a composition comprises acrosslinked lubricin where the crosslinked lubricin represents, e.g.,about 1% by weight, about 2% by weight, about 3% by weight, about 4% byweight, about 5% by weight, about 6% by weight, about 7% by weight,about 8% by weight, or about 9%, or about 10% by weight, of the totallubricin present in the composition. In other aspects of thisembodiment, a composition comprises a crosslinked lubricin where thecrosslinked lubricin represents, e.g., at most 1% by weight, at most 2%by weight, at most 3% by weight, at most 4% by weight, at most 5% byweight, at most 6% by weight, at most 7% by weight, at most 8% byweight, at most 9% by weight, or at most 10% by weight, of the totallubricin present in the composition. In yet other aspects of thisembodiment, a composition comprises a crosslinked lubricin where thecrosslinked lubricin represents, e.g., about 0% to about 10% by weight,about 1% to about 10% by weight, about 3% to about 10% by weight, orabout 5% to about 10% by weight, of the total lubricin present in thecomposition.

In other aspects of this embodiment, a composition comprises acrosslinked lubricin where the degree of crosslinking is about 1%, about2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%.In yet other aspects of this embodiment, a composition comprises acrosslinked lubricin where the degree of crosslinking is at most 1%, atmost 2%, at most 3%, at most 4%, at most 5%, at most 6%, at most 7%, atmost 8%, at most 9%, at most 10%, at most 11%, at most 12%, at most 13%,at most 14%, or at most 15%. in still other aspects of this embodiment,a composition comprises a crosslinked lubricin where the degree ofcrosslinking is about 1% to about 15%, about 2% to about 11%, about 3%to about 10%, about 1% to about 5%, about 10% to about 15%, about 11% toabout 15%, about 6% to about 10%, or about 6% to about 8%.

In another embodiment, a composition comprises a crosslinkedpolysaccharide. In aspect of this embodiment, a composition comprises acrosslinked dextran, a crosslinked dextrin, a crosslinked starch, acrosslinked hetastarch, a crosslinked glycogen, a crosslinked polyvinylacetate, a crosslinked polyvinyl pyrrolidone, a crosslinked polyethyleneglycol, a crosslinked polyethylene imine, a crosslinked cellulose, acrosslinked methyl cellulose, a crosslinked carboxymethyl cellulose, acrosslinked hydroxyethyl cellulose, a crosslinked hydroxypropylcellulose, a crosslinked hydroxyethyl methyl cellulose, or a crosslinkedhydroxypropyl methyl cellulose. In other aspects of this embodiment, acomposition comprises a crosslinked polysaccharide where the crosslinkedpolysaccharide represents, e.g., about 1% by weight, about 2% by weight,about 3% by weight, about 4% by weight, about 5% by weight, about 6% byweight, about 7% by weight, about 8% by weight, or about 9%, or about10% by weight, of the total polysaccharide present in the composition.In yet other aspects of this embodiment, a composition comprises acrosslinked polysaccharide where the crosslinked polysacchariderepresents, e.g., at most 1% by weight, at most 2% by weight, at most 3%by weight, at most 4% by weight, at most 5% by weight, at most 6% byweight, at most 7% by weight, at most 8% by weight, at most 9% byweight, or at most 10% by weight, of the total polysaccharide present inthe composition. In still other aspects of this embodiment, acomposition comprises a crosslinked polysaccharide where the crosslinkedpolysaccharide represents, e.g., about 0% to about 10% by weight, about1% to about 10% by weight, about 3% to about 10% by weight, or about 5%to about 10% by weight, of the total polysaccharide present in thecomposition.

In other aspects of this embodiment, a composition comprises acrosslinked polysaccharide where the degree of crosslinking is about 1%,about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%,about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, orabout 15%. In yet other aspects of this embodiment, a compositioncomprises a crosslinked polysaccharide where the degree of crosslinkingis at most 1%, at most 2%, at most 3%, at most 4%, at most 5%, at most6%, at most 7%, at most 8%, at most 9%, at most 10%, at most 11%, atmost 12%, at most 13%, at most 14%, or at most 15%. In still otheraspects of this embodiment, a composition comprises a crosslinkedpolysaccharide where the degree of crosslinking is about 1% to about15%, about 2% to about 11%, about 3% to about 10%, about 1% to about 5%,about 10% to about 15%, about 11% to about 15%, about 6% to about 10%,or about 6% to about 8%.

Aspects of the present specification provide, in part, a compositioncomprising an uncrosslinked matrix polymer. As used herein, the term“uncrosslinked” refers to a lack of intermolecular bonds joining theindividual matrix polymer molecules, or monomer chains. As such, anuncrosslinked matrix polymer is not linked to any other matrix polymerby an intermolecular bond.

Aspects of the present specification provide, in part, a compositioncomprising a substantially uncrosslinked matrix polymer. As sued herein,the term “substantially uncrosslinked” refers to the presence ofuncrosslinked matrix polymers in a composition disclosed herein at alevel of at least 90% by weight of the composition, with the remainingat most 10% by weight of the composition being comprised of othercomponents including crosslinked matrix polymers.

In an embodiment, a composition comprises a substantially uncrosslinkedmatrix polymer. In other aspects of this embodiment, a compositioncomprises an uncrosslinked matrix polymer where the uncrosslinked matrixpolymer represents, e.g., about 90% by weight, about 91% by weight,about 92% by weight, about 93% by weight, about 94% by weight, about 95%by weight, about 96% by weight, about 97% by weight, about 98% byweight, or about 99%, or about 100% by weight, of the total matrixpolymer present in the composition. In yet other aspects of thisembodiment, a composition comprises an uncrosslinked matrix polymerwhere the uncrosslinked matrix polymer represents, e.g., at least 90% byweight, at least 91% by weight, at least 92% by weight, at least 93% byweight, at least 94% by weight, at least 95% by weight, at least 96% byweight, at least 97% by weight, at least 98% by weight, or at least 99%by weight, of the total matrix polymer present in the composition. Instill other aspects of this embodiment, a composition comprises anuncrosslinked matrix polymer where the uncrosslinked matrix polymerrepresents, e.g., about 90% to about 100% by weight, about 93% to about100% by weight, about 95% to about 100% by weight, or about 97% to about100% by weight, of the total matrix polymer present in the composition.

In another embodiment, a composition comprises a substantiallyuncrosslinked glycosaminoglycan. In aspects of this embodiment, acomposition comprises a substantially uncrosslinked chondroitin sulfatepolymer, a substantially uncrosslinked dermatan sulfate polymer, asubstantially uncrosslinked keratan sulfate polymer, a substantiallyuncrosslinked heparan polymer, a substantially uncrosslinked heparansulfate polymer, or a substantially uncrosslinked hyaluronan polymer. Inother aspects of this embodiment, a fluid composition comprises anuncrosslinked glycosaminoglycan where the uncrosslinkedglycosaminoglycan represents, e.g., about 90% or more by weight, about91% or more by weight, about 92% or more by weight, about 93% or more byweight, about 94% or more by weight, about 95% or more by weight, about96% or more by weight, about 97% or more by weight, about 98% or more byweight, or about 99% or more, or about 100% by weight, of the totalglycosaminoglycan present in the composition. In yet other aspects ofthis embodiment, a fluid composition comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan represents,e.g., about 90% to about 100% by weight, about 93% to about 100% byweight, about 95% to about 100% by weight, or about 97% to about 100% byweight, of the total glycosaminoglycan present in the composition.

In yet another embodiment, a fluid composition comprises a substantiallyuncrosslinked lubricin. In aspects of this embodiment, a fluidcomposition comprises an uncrosslinked lubricin where the uncrosslinkedlubricin represents, e_g., about 90% or more by weight, about 91% ormore by weight, about 92% or more by weight, about 93% or more byweight, about 94% or more by weight, about 95% or more by weight, about96% or more by weight, about 97% or more by weight, about 98% or more byweight, or about 99% or more, or about 100% by weight, of the totallubricin present in the composition. In other aspects of thisembodiment, a fluid composition comprises an uncrosslinked lubricinwhere the uncrosslinked lubricin represents, e.g., about 90% to about100% by weight, about 93% to about 100% by weight, about 95% to about100% by weight, or about 97% to about 100% by weight, of the totallubricin present in the composition.

In another embodiment, a composition comprises a substantiallyuncrosslinked polysaccharide. In aspects of this embodiment, acomposition comprises a substantially uncrosslinked dextran, asubstantially uncrosslinked dextrin, a substantially uncrosslinkedstarch, a substantially uncrosslinked hetastarch, a substantiallyuncrosslinked glycogen, a substantially uncrosslinked polyvinyl acetate,a substantially uncrosslinked polyvinyl pyrrolidone, a substantiallyuncrosslinked polyethylene glycol, a substantially uncrosslinkedpolyethylene imine, a substantially uncrosslinked cellulose, asubstantially uncrosslinked methyl cellulose, a substantiallyuncrosslinked carboxymethyl cellulose, a substantially uncrosslinkedhydroxyethyl cellulose, a substantially uncrosslinked hydroxypropylcellulose, a substantially uncrosslinked hydroxyethyl methyl cellulose,or a substantially uncrosslinked hydroxypropyl methyl cellulose. Inother aspects of this embodiment, a composition comprises anuncrosslinked polysaccharide where the uncrosslinked polysacchariderepresents, e.g., about 90% or more by weight, about 91% or more byweight, about 92% or more by weight, about 93% or more by weight, about94% or more by weight, about 95% or more by weight, about 96% or more byweight, about 97% or more by weight, about 98% or more by weight, orabout 99% or more, or about 100% by weight, of the total polysaccharidepresent in the composition. in yet other aspects of this embodiment, acomposition comprises an uncrosslinked polysaccharide where theuncrosslinked polysaccharide represents, e.g., about 90% to about 100%by weight, about 93% to about 100% by weight, about 95% to about 100% byweight, or about 97% to about 100% by weight, of the totalpolysaccharide present in the composition.

Aspects of the present specification provide, in part, a compositionthat is essentially free of a crosslinked matrix polymer. As usedherein, the term “essentially free” (or “consisting essentially of”)refers to a composition where only trace amounts of cross-linked matrixpolymers can be detected.

In an embodiment, a composition comprises a glycosaminoglycan that isessentially free of a crosslinked glycosaminoglycan. In an aspect ofthis embodiment, a composition comprises a chondroitin sulfate that isessentially free of a crosslinked chondroitin sulfate polymer, adermatan sulfate essentially free of a crosslinked dermatan sulfatepolymer, a keratan sulfate essentially free of a crosslinked keratansulfate polymer, a heparan essentially free of a crosslinked heparanpolymer, a heparan sulfate essentially free of a crosslinked heparansulfate polymer, or a hyaluronan sulfate essentially free of acrosslinked hyaluronan polymer.

In yet another embodiment, a fluid composition comprises a lubricin thatis essentially free of a crosslinked lubricin.

In an embodiment, a composition comprises a polysaccharide that isessentially free of a crosslinked polysaccharide. In an aspect of thisembodiment, a composition comprises a dextran that is essentially freeof a crosslinked dextran, dextrin that is essentially free of acrosslinked dextrin, dextran that is essentially free of a crosslinkedstarch, hetastarch that is essentially free of a crosslinked hetastarch,glycogen that is essentially free of a crosslinked glycogen, polyvinylacetate that is essentially free of a crosslinked polyvinyl acetate,polyvinyl pyrrolidone that is essentially free of a crosslinkedpolyvinyl pyrrolidone, polyethylene glycothat is essentially free of acrosslinked polyethylene glycol, polyethylene imine that is essentiallyfree of a crosslinked polyethylene imine, cellulose that is essentiallyfree of a crosslinked cellulose, methyl cellulose that is essentiallyfree of a crosslinked methyl cellulose, carboxymethyl cellulose that isessentially free of a crosslinked carboxymethyl cellulose, hydroxyethylcellulose that is essentially free of a crosslinked hydroxyethylcellulose, hydroxypropyl cellulose that is essentially free of acrosslinked hydroxypropyl cellulose, hydroxyethyl methyl cellulosthat isessentially free of a crosslinked hydroxyethyl methyl cellulose, orhydroxypropyl methyl cellulose that is essentially free of a crosslinkedhydroxypropyl methyl cellulose.

Aspects of the present specification provide, in part, a compositionthat is entirely free of a crosslinked matrix polymer. As used herein,the term “entirely free” refers to a fluid composition that within thedetection range of the instrument or process being used, crosslinkedmatrix polymers cannot be detected or its presence cannot be confirmed.

In an embodiment, a composition comprises a glycosaminoglycan that isentirely free of a crosslinked glycosaminoglycan. In an aspect of thisembodiment, a composition comprises a chondroitin sulfate that isentirely free of a crosslinked chondroitin sulfate polymer, a dermatansulfate entirely free of a crosslinked dermatan sulfate polymer, akeratan sulfate entirely free of a crosslinked keratan sulfate polymer,a heparan entirely free of a crosslinked heparan polymer, a heparansulfate entirely free of a crosslinked heparan sulfate polymer, or ahyaluronan sulfate entirely free of a crosslinked hyaluronan polymer.

In yet another embodiment, a fluid composition comprises a lubricin thatis entirely free of a crosslinked lubricin.

In an embodiment, a composition comprises a polysaccharide that isentirely free of a crosslinked polysaccharide. In an aspect of thisembodiment, a composition comprises a dextran that is entirely free of acrosslinked dextran, dextrin that is entirely free of a crosslinkeddextrin, dextran that is entirely free of a crosslinked starch,hetastarch that is entirely free of a crosslinked hetastarch, glycogenthat is entirely free of a crosslinked glycogen, polyvinyl acetate thatis entirely free of a crosslinked polyvinyl acetate, polyvinylpyrrolidone that is entirely free of a crosslinked polyvinylpyrrolidone, polyethylene glycothat is entirely free of a crosslinkedpolyethylene glycol, polyethylene imine that is entirely free of acrosslinked polyethylene imine, cellulose that is entirely free of acrosslinked cellulose, methyl cellulose that is entirely free of acrosslinked methyl cellulose, carboxymethyl cellulose that is entirelyfree of a crosslinked carboxymethyl cellulose, hydroxyethyl cellulosethat is entirely free of a crosslinked hydroxyethyl cellulose,hydroxypropyl cellulose that is entirely free of a crosslinkedhydroxypropyl cellulose, hydroxyethyl methyl cellulosthat is entirelyfree of a crosslinked hydroxyethyl methyl cellulose, or hydroxypropylmethyl cellulose that is entirely free of a crosslinked hydroxypropylmethyl cellulose.

Aspects of the present specification provide, in part, a compositioncomprising a ratio of crosslinked matrix polymer and uncrosslinkedpolymer. This ratio of crosslinked and uncrosslinked matrix polymer isalso known as the gel:fluid ratio. Any gel:fluid ratio is useful inmaking the compositions disclosed herein with the proviso that suchratio produces a composition disclosed herein that improves a skincondition as disclosed herein. Non-limiting examples of gel:fluid ratiosinclude 100:0, 98:2, 90:10, 75:25, 70:30, 60:40, 50:50, 40:60, 30:70,25:75, 10:90; 2:98, and 0:100.

In an embodiment, a composition comprises a crosslinked matrix polymerand an uncrosslinked matrix polymer. In another aspect of thisembodiment, a composition comprises a crosslinked matrix polymer and anuncrosslinked matrix polymer where the gel:fluid ratio is sufficient toform a fluid. In other aspects of this embodiment, a compositioncomprises a crosslinked matrix polymer and an uncrosslinked matrixpolymer where the gel:fluid ratio is, e.g., about 0:100, about 1:99,about 2:98, about 3:97, about 4:96, about 5:95, about 6:94, about 7:93,about 8:92, about 9:91, or about 10:90. In yet other aspects of thisembodiment, a composition comprises a crosslinked matrix polymer and anuncrosslinked matrix polymer where the gel:fluid ratio is, e.g., at most1:99, at most 2:98, at most 3:97, at most 4:96, at most 5:95, at most6:94, at most 7.:93, at most 8:92, at most 9:91, or at most 10:90. Instill other aspects of this embodiment, a composition comprises acrosslinked matrix polymer and an uncrosslinked matrix polymer where thegel:fluid ratio is, e.g., about 0:100 to about 3:97, about 0:100 toabout 5:95, or about 0:100 to about 10:90.

In other aspects of this embodiment, a composition comprises acrosslinked matrix polymer and an uncrosslinked matrix polymer where thegel:fluid ratio is, e.g., about 15:85, about 20:80, about 25:75, about30:70, about 35:65, about 40:60, about 45:55, about 50:50, about 55:45,about 60:40, about 65:35, about 70:30, about 75:25, about 80:20, about85:15, about 90:10, about 95:5, about 98:2, or about 100:0. In yet otheraspects of this embodiment, a composition comprises a crosslinked matrixpolymer and an uncrosslinked matrix polymer where the gel:fluid ratiois, e.g., at most 15:85, at most 20:80, at most 25:75, at most 30:70, atmost 35:65, at most 40:60, at most 45:55, at most 50:50, at most 55:45,at most 60:40, at most 65:35, at most 70:30, at most 75:25, at most80:20, at most 85:15, at most 90:10, at most 95:5, at most 98:2, or atmost 100:0. In still other aspects of this embodiment, a compositioncomprises a crosslinked matrix polymer and an uncrosslinked matrixpolymer where the gel:fluid ratio is, e.g., about 10:90 to about 70:30,about 15:85 to about 70:30, about 10:90 to about 55:45, about 80:20 toabout 95:5, about 90:10 to about 100:0, about 75:25 to about 100:0, orabout 60:40 to about 100:0.

In still another embodiment, a composition comprises an uncrosslinkedmatrix polymer where the uncrosslinked matrix polymer is present in anamount sufficient to improve a condition of the skin, such as, e.g.,hydration or elasticity. In aspects of this embodiment, a compositioncomprises an uncrosslinked matrix polymer where the uncrosslinked matrixpolymer is present at a concentration of, e.g., about 5 mg/mL, about 6mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL,about 11 mg/mL, about 12 mg/mL, about 13 mg/mL, about 13.5 mglmL, about14 mg/mL, about 15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18mg/mL, about 19 mg/mL, or about 20 mg/mL. In other aspects of thisembodiment, a composition comprises an uncrosslinked matrix polymerwhere the uncrosslinked matrix polymer is present at a concentration of,e.g., at least 1 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 15mg/mL, at least 20 mg/mL, or at least 25 mg/mL. In yet other aspects ofthis embodiment, a composition comprises an uncrosslinked matrix polymerwhere the uncrosslinked matrix polymer is present at a concentration of,e.g., at most 1 mg/mL, at most 5 mg/mL, at most 10 mg/mL, at most 15mg/mL, at most 20 mg/mL, or at most 25 mg/mL. In still other aspects ofthis embodiment, a composition comprises an uncrosslinked matrix polymerwhere the uncrosslinked matrix polymer is present at a concentration of,e.g., about 7.5 mg/mL to about 19.5 mg/m L, about 8.5 mg/mL to about18.5 mg/mL, about 9.5 mg/mL to about 17.5 mg/mL, about 10.5 mg/mL toabout 16.5 mglmL, about 11.5 mg/mL to about 15.5 mg/mL, or about 12.5mg/mL to about 14.5 mg/mL.

In aspects of this embodiment, a composition comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan is presentat a concentration of, e.g., about 2 mg/mL, about 3 mg/mL, about 4mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about9 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, about 13 mg/mL,about 13.5 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL, about17 mg/mL, about 18 mg/mL, about 19 mg/mL, or about 20 mg/mL. In otheraspects of this embodiment, a composition comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan is presentat a concentration of, e.g., at least 1 mg/mL, at least 2 mg/mL, atleast 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 10 mg/mL, atleast 15 mg/m L, at least 20 mg/mL, or at least 25 mg/mL. In yet otheraspects of this embodiment, a composition comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan is presentat a concentration of, e.g., at most 1 mg/mL, at most 2 mg/mL, at most 3mg/m L, at most 4 mg/mL, at most 5 mg/mL, at most 10 mg/mL, at most 15mg/mL, at most 20 mg/mL, or at most 25 mg/mL. In still other aspects ofthis embodiment, a composition comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan is presentat a concentration of, e.g., about 7.5 mg/mL to about 19.5 mg/mL, about8.5 mg/mL to about 18.5 mg/mL, about 9.5 mg/mL to about 17.5 mg/mL,about 10.5 mg/mL to about 16.5 mg/mL, about 11.5 mg/mL to about 15.5mg/mL, or about 12.5 mg/mL to about 14.5 mg/mL.

In aspects of this embodiment, a composition comprises an uncrosslinkedlubricin where the uncrosslinked lubricin is present at a concentrationof, e.g., about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL,about 9 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, about 13mg/mL, about 13.5 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL,about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, or about 20 mg/mL. Inother aspects of this embodiment, a composition comprises anuncrosslinked lubricin where the uncrosslinked lubricin is present at aconcentration of, e.g., at least 1 mg/mL, at least 5 mg/mL, at least 10mg/mL, at least 15 mg/mL, at least 20 mg/mL, or at least 25 mg/mL. Inyet other aspects of this embodiment, a composition comprises anuncrosslinked lubricin where the uncrosslinked lubricin is present at aconcentration of, e.g., at most 1 mg/mL, at most 5 mg/mL, at most 10mg/mL, at most 15 mg/mL, at most 20 mg/mL, or at most 25 mg/mL. In stillother aspects of this embodiment, a composition comprises anuncrosslinked lubricin where the uncrosslinked lubricin is present at aconcentration of, e.g., about 7.5 mg/mL to about 19.5 mg/mL, about 8.5mg/mL to about 18.5 mg/mL, about 9.5 mg/mL to about 17.5 mg/mL, about10.5 mg/mL to about 16.5 mg/mL, about 11.5 mg/mL to about 15.5 mg/mL, orabout 12.5 mg/mL to about 14.5 mg/mL.

In aspects of this embodiment, a composition comprises an uncrosslinkedpolysaccharide where the uncrosslinked polysaccharide is present at aconcentration of, e.g., about 2 mg/mL, about 3 mg/mL, about 4 mg/mL,about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL, about 9mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, about 13 mg/mL,about 13.5 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL, about17 mg/mL, about 18 mg/mL, about 19 mg/mL, or about 20 mg/mL. In otheraspects of this embodiment, a composition comprises an uncrosslinkedpolysaccharide where the uncrosslinked polysaccharide is present at aconcentration of, e.g., at least 1 mg/mL, at least 2 mg/mL, at least 3mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least15 mg/mL, at least 20 mg/mL, or at least 25 mg/mL. In yet other aspectsof this embodiment, a composition comprises an uncrosslinkedpolysaccharide where the uncrosslinked polysaccharide is present at aconcentration of, e.g., at most 1 mg/mL, at most 2 mg/mL, at most 3mg/mL, at most 4 mg/mL, at most 5 mg/mL, at most 10 mg/mL, at most 15mg/mL, at most 20 mg/mL, or at most 25 mg/mL. In still other aspects ofthis embodiment, a composition comprises an uncrosslinked polysaccharidewhere the uncrosslinked polysaccharide is present at a concentration of,e.g., about 7.5 mg/mL to about 19.5 mg/mL, about 8.5 mg/mL to about 18.5mg/mL, about 9.5 mg/mL to about 17.5 mg/mL, about 10.5 mg/mL to about16.5 mg/mL, about 11.5 mg/mL to about 15.5 mg/mL, or about 12.5 mg/mL toabout 14.5 mg/mL.

In an embodiment, a composition comprises an uncrosslinked hyaluronanwhere the uncrosslinked hyaluronan comprises a combination of both highmolecular weight hyaluronan and low molecular weight hyaluronan in aratio of about 20:1, about 15:1, about 10:1, about 5:1, about 1:1, about1:5 about 1:10, about 1:15, or about 1:20.

In another embodiment, a composition comprises an uncrosslinkedhyaluronan where the uncrosslinked hyaluronan comprises a combination ofboth high molecular weight hyaluronan and low molecular weighthyaluronan, in various ratios. As used herein, the term “high molecularweight hyaluronan” refers to a hyaluronan polymer that has a molecularweight of 1,000,000 Da or greater. Non-limiting examples of a highmolecular weight hyaluronan include a hyaluronan of about 1,500,000 Da,a hyaluronan of about 2,000,000 Da, a hyaluronan of about 2,500,000 Da,a hyaluronan of about 3,000,000 Da, a hyaluronan of about 3,500,000 Da,a hyaluronan of about 4,000,000 Da, a hyaluronan of about 4,500,000 Da,and a hyaluronan of about 5,000,000 Da. As used herein, the term “lowmolecular weight hyaluronan” refers to a hyaluronan polymer that has amolecular weight of less than 1,000,000 Da. Non-limiting examples of alow molecular weight hyaluronan include a hyaluronan of about 200,000Da, a hyaluronan of about 300,000 Da, a hyaluronan of about 400,000 Da,a hyaluronan of about 500,000 Da, a hyaluronan of about 600,000 Da, ahyaluronan of about 700,000 Da, a hyaluronan of about 800,000 Da, and ahyaluronan of about 900,000 Da.

In other aspects of this embodiment, a composition comprises acrosslinked hyaluronan where the crosslinked hyaluronan has a meanmolecular weight of, e.g., about 1,000,000 Da, about 1,500,000 Da, about2,000,000 Da, about 2,500,000 Da, about 3,000,000 Da, about 3,500,000Da, about 4,000,000 Da, about 4,500,000 Da, or about 5,000,000 Da. Inyet other aspects of this embodiment, a composition comprises acrosslinked hyaluronan where the crosslinked hyaluronan has a meanmolecular weight of, e.g., at least 1,000,000 Da, at least 1,500,000 Da,at least 2,000,000 Da, at least 2,500,000 Da, at least 3,000,000 Da, atleast 3,500,000 Da, at least 4,000,000 Da, at least 4,500,000 Da, or atleast 5,000,000 Da. In still other aspects of this embodiment, acomposition comprises a crosslinked hyaluronan where the crosslinkedhyaluronan has a mean molecular weight of, e.g., about 1,000,000 Da toabout 5,000,000 Da, about 1,500,000 Da to about 5,000,000 Da, about2,000,000 Da to about 5,000,000 Da, about 2,500,000 Da to about5,000,000 Da, about 2,000,000 Da to about 3,000,000 Da, about 2,500,000Da to about 3,500,000 Da, or about 2,000,000 Da to about 4,000,000 Da.

In other aspects of this embodiment, a composition comprises anuncrosslinked hyaluronan where the uncrosslinked hyaluronan has a meanmolecular weight of, e.g., about 1,000,000 Da, about 1,500,000 Da, about2,000,000 Da, about 2,500,000 Da, about 3,000,000 Da, about 3,500,000Da, about 4,000,000 Da, about 4,500,000 Da, or about 5,000,000 Da. Inyet other aspects of this embodiment, a composition comprises anuncrosslinked hyaluronan where the uncrosslinked hyaluronan has a meanmolecular weight of, e.g., at least 1,000,000 Da, at least 1,500,000 Da,at least 2,000,000 Da, at least 2,500,000 Da, at least 3,000,000 Da, atleast 3,500,000 Da, at least 4,000,000 Da, at least 4,500,000 Da, or atleast 5,000,000 Da. In still other aspects of this embodiment, acomposition comprises an uncrosslinked hyaluronan where theuncrosslinked hyaluronan has a mean molecular weight of, e.g., about1,000,000 Da to about 5,000,000 Da, about 1,500,000 Da to about5,000,000 Da, about 2,000,000 Da to about 5,000,000 Da, about 2,500,000Da to about 5,000,000 Da, about 2,000,000 Da to about 3,000,000 Da,about 2,500,000 Da to about 3,500,000 Da, or about 2,000,000 Da to about4,000,000 Da. In further aspects, a composition comprises anuncrosslinked hyaluronan where the uncrosslinked hyaluronan has a meanmolecular weight of, e.g., greater than 2,000,000 Da and less than about3,000,000 Da, greater than 2,000,000 Da and less than about 3,500,000Da, greater than 2,000,000 Da and less than about 4,000,000 Da, greaterthan 2,000,000 Da and less than about 4,500,000 Da, greater than2,000,000 Da and less than about 5,000,000 Da.

A composition disclosed herein comprises a gel phase including a silkfibroin hydrogel component or particle and matrix polymer hydrogelcomponent or particle. In aspects of this embodiment, the percent amountof silk fibroin hydrogel present in a composition relative to matrixpolymer hydrogel is from about 0.1% (v/v) to about 25% (v/v). In aspectsof this embodiment, the percent amount of matrix polymer hydrogelpresent in a composition relative to silk fibroin hydrogel is from about99.9% (v/v) to about 75% (v/v). In aspects of this embodiment, the ratioof silk fibroin hydrogel to matrix polymer hydrogel in the gel phase ofa composition comprises, e.g., about 0.1% (v/v) silk fibroin hydrogeland about 99.9% (v/v) matrix polymer hydrogel, about 1% (v/v) silkfibroin hydrogel and about 99% (v/v) matrix polymer hydrogel, about 5%(v/v) silk fibroin hydrogel and about 95% (v/v) matrix polymer hydrogel,about 10% (v/v) silk fibroin hydrogel and about 90% (v/v) matrix polymerhydrogel, about 15% (v/v) silk fibroin hydrogel and about 85% (v/v)matrix polymer hydrogel, about 20% (v/v) silk fibroin hydrogel and about80% (v/v) matrix polymer hydrogel, or about 25% (v/v) silk fibroinhydrogel and about 75% (v/v) matrix polymer hydrogel.

A composition disclosed herein may comprise a gel phase where the silkfibroin hydrogel component and matrix polymer hydrogel component areprocessed separately. The resulting processed hydrogel materials, e.g.,hydrogel particles of both types, are then mixed together, such as,e.g., after a milling step and/or after re-homogenization in a carrierphase, to form the final composition. In addition, a matrix polymer maybe initially mixed with depolymerized silk fibroin solution, withsubsequent polymerization occurring only after the completion of themixing step to form an integrated matrix polymer/silk fibroin compositehydrogel. Similarly, the silk fibroin and matrix polymers may be linkedtogether to form a hydrogel composite that is then subsequentlyprocessed into the gel phase of the composition. Such linkage can occurby a typical crosslinking method or by linking the matrix polymer to thesilk fibroin hydrogel via a peptide linker disclosed herein, such as,e.g., a five-amino acid peptide “tail” and synthetic molecule. Asdisclosed herein, a composition may comprise a gel phase that comprisesboth separately processed hydrogel components as well as particles ofhydrogel composites.

As a non-limiting example, a solution comprising about 1% to about 30%depolymerized silk fibroin may be mixed with about 6 mg/g to about 30mg/g of hyaluronan having a degree of crosslinking of from 0 to about17% where the percent weight of the silk fibroin component is from about1% to about 75%. As another non-limiting example, hydrogel particlescomprising from about 1% to about 8% silk fibroin are mixed withhydrogel particles comprising about 6 mg/g to about 30 mg/g ofhyaluronan having a degree of crosslinking of from 0 to about 17% wherethe percent weight of the silk fibroin component is from about 1% toabout 75%. As yet another non-limiting example, a hydrogel compositioncomprising hydrogel particles comprising from about 1% to about 8% silkfibroin mixed together with a carrier phase (about 20% (v/v) to about50% (v/v)) is mixed with a hydrogel composition comprising hydrogelparticles comprising about 6mg/g to about 30 mg/g of hyaluronan having adegree of crosslinking of from 0 to about 17% where the percent weightof the silk fibroin component is from about 1% to about 75%.

Aspects of the present specification provide, in part, a compositioncomprising a silk fibroin hydrogel component or particle and matrixpolymer hydrogel component or particle having an opacity. Opacity is themeasure of impenetrability to electromagnetic or other kinds ofradiation, especially visible light. An opaque object is neithertransparent (allowing all light to pass through) nor translucent(allowing some light to pass through). In certain applications, it wouldbe an advantage to have an opaque composition. For example, inapplications where a composition disclosed herein is administered to asuperficial region, an opaque composition provides coloration andappearance of the overlying skin.

In an embodiment, a composition comprising a silk fibroin hydrogel and apolymer matrix is optically opaque. In aspects of this embodiment, acomposition comprising a silk fibroin hydrogel and a polymer matrixtransmits, e.g., about 5% of the light, about 10% of the light, about15% of the light, about 20% of the light, about 25% of the fight, about30% of the light, about 35% of the light, about 40% of the light, about45% of the light, about 50% of the light, about 55% of the light, about60% of the light, about 65% of the light, or about 70% of the light. Inother aspects of this embodiment, a composition comprising a silkfibroin hydrogel and a polymer matrix transmits, e.g., at most 5% of thelight, at most 10% of the light, at most 15% of the light, at most 20%of the light, at most 25% of the light, at most 30% of the light, atmost 35% of the light, at most 40% of the light, at most 45% of thelight, at most 50% of the light, at most 55% of the light, at most 60%of the light, at most 65% of the light, at most 70% of the light, or atmost 75% of the light. In other aspects of this embodiment, acomposition comprising a silk fibroin hydrogel and a polymer matrixtransmits, e.g., about 5% to about 15%, about 5% to about 20%, about 5%to about 25%, about 5% to about 30%, about 5% to about 35%, about 5% toabout 40%, about 5% to about 45%, about 5% to about 50%, about 5% toabout 55%, about 5% to about 60%, about 5% to about 65%, about 5% toabout 70%, about 5% to about 75%, about 15% to about 20%, about 15% toabout 25%, about 15% to about 30%, about 15% to about 35%, about 15% toabout 40%, about 15% to about 45%, about 15% to about 50%, about 15% toabout 55%, about 15% to about 60%, about 15% to about 65%, about 15% toabout 70%, about 15% to about 75%, about 25% to about 35%, about 25% toabout 40%, about 25% to about 45%, about 25% to about 50%, about 25% toabout 55%, about 25% to about 60%, about 25% to about 65%, about 25% toabout 70%, or about 25% to about 75%, of the light.

In aspects of this embodiment, a composition comprising a silk fibroinhydrogel and a polymer matrix exhibits, e.g., about 5%, about 10%, about15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%,about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, about 100% reduction intyndalling. In other aspects of this embodiment, a compositioncomprising a silk fibroin hydrogel and a polymer matrix, e.g., at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 100%,reduction in tyndalling. In other aspects of this embodiment, acomposition comprising a silk fibroin hydrogel and a polymer matrixexhibits, e.g., about 20% to about 100%, about 50% to about 100%, about70% to about 100%, about 15% to about 35%, about 20% to about 40%, about25% to about 45%, about 30% to about 50%, about 35% to about 55%, about40% to about 60%, about 45% to about 65%, about 50% to about 70%, about55% to about 75%, about 60% to about 80%, about 65% to about 85%, about70% to about 90%, about 75% to about 95%, or about 80% to about 100%,reduction in tyndalling.

Aspects of the present specification provide, in part, a compositioncomprising a carrier phase. A composition disclosed herein may, but maynot, include a carrier phase. As such, the disclosed compositions can bemonophasic or multiphasic compositions. As used herein, the term“carrier phase” is synonymous with “carrier” and refers to a materialused to increase fluidity of a hydrogel. A carrier is advantageously aphysiologically-acceptable carrier and may include one or moreconventional excipients useful in pharmaceutical compositions. As usedherein, the term “a physiologically-acceptable carrier” refers to acarrier in accord with, or characteristic of, the normal functioning ofa living organism. As such, administration of a composition comprising ahydrogel and a carrier has substantially no long term or permanentdetrimental effect when administered to a mammal. The presentcompositions include a carrier where a major of the volume is water orsaline. However, other useful carriers include any physiologicallytolerable material which improves upon extrudability or intrudability ofthe hydrogel through a needle or into a target host environment.Potential carriers could include but are not limited to physiologicalbuffer solutions, serum, other protein solutions, gels composed ofpolymers including proteins, glycoproteins, proteoglycans, orpolysaccharides. Any of the indicated potential carriers may be eithernaturally derived, wholly synthetic, or combinations of both.

The volume of carrier per volume of hydrogel may be increased ordecreased in a range between 0% to about 100% depending upon the desiredphysical properties of the resultant composition including dosedelivery, viscosity, injectability, and desired in vivo behavioralcharacteristics. This carrier is then mixed with the hydrogel untilachieving a “uniform” consistency which may be termed an emulsion orsuspension. More specifically, for example, a hydrogel may be passedthrough an 18 g needle several times to create hydrogel particles,injecting back and forth between a pair of syringes, then this procedurerepeated with 22 g needles affixed to 1 mL syringes. Advantages derivedfrom adding a carrier to a hydrogel or hydrogel particles includedecreased viscosity in the extracellular in vivo microenvironment;release of focal mechanical stress loading after drug delivery platformadministration; and improved ionic composition resulting in improvedbiocompatibility.

Aspects of the present specification provide, in part, a compositiondisclosed herein exhibiting a dynamic viscosity. Viscosity is resistanceof a fluid to shear or flow caused by either shear stress or tensilestress. Viscosity describes a fluid's internal resistance to flow causedby intermolecular friction exerted when layers of fluids attempt toslide by one another and may be thought of as a measure of fluidfriction. The less viscous the fluid, the greater its ease of movement(fluidity).

Viscosity can be defined in two ways; dynamic viscosity (μ, although ηis sometimes used) or kinematic viscosity (v). Dynamic viscosity, alsoknown as absolute or complex viscosity, is the tangential force per unitarea required to move one horizontal plane with respect to the other atunit velocity when maintained a unit distance apart by the fluid. The SIphysical unit of dynamic viscosity is the Pascal-second (Pa·s), which isidentical to N·m−2·s. Dynamic viscosity can be expressed as τ=μdvx/dz,where τ=shearing stress, μ=dynamic viscosity, and dvx/dz is the velocitygradient over time. For example, if a fluid with a viscosity of one Pa·sis placed between two plates, and one plate is pushed sideways with ashear stress of one Pascal, it moves a distance equal to the thicknessof the layer between the plates in one second. Dynamic viscositysymbolize by is also used, is measured with various types of rheometers,devices used to measure the way in which a liquid, suspension or slurryflows in response to applied forces.

Kinematic viscosity (v) is the ratio of dynamic viscosity to density, aquantity in which no force is involved and is defined as follows: v=μ/ρ,where μ is the dynamic viscosity ρ is density with the SI unit of kg/m³.Kinematic viscosity is usually measured by a glass capillary viscometeras has an SI unit of m²/s.

The viscosity of a fluid is highly temperature dependent and for eitherdynamic or kinematic viscosity to be meaningful, the referencetemperature must be quoted. For the viscosity values disclosed herein, adynamic viscosity is measured at 1 Pa with a cone/plane geometry 2°/40cm and a temperature of 20° C. Examples of the dynamic viscosity ofvarious fluids at 20° C. is as follows: water is about 1.0×10⁻³ Pa·s,blood is about 3-4×10^(')Pa·s, vegetable oil is about 60-85×10⁻³ Pa·s,motor oil SE 30 is about 0.2 Pa·s, glycerin is about 1.4 Pa·s, maplesyrup is about 2-3 Pa·s, honey is about 10 Pa·s, chocolate syrup isabout 10-25 Pa·s, peanut butter is about 150-250 Pa·s, lard is about1,000 Pa·s, vegetable shortening is about 1,200 Pa·s, and tar is about30,000 Pa·s.

In aspects of this embodiment, a composition disclosed herein exhibits adynamic viscosity of, e.g., about 10 Pa·s, about 20 Pa·s, about 30 Pa·s,about 40 Pa·s, about 50 Pa·s, about 60 Pa·s, about 70 Pa·s, about 80Pa·s, about 90 Pa·s, about 100 Pa·s, about 125 Pa·s, about 150 Pa·s,about 175 Pa·s, about 200 Pa·s, about 225 Pa·s, about 250 Pa·s, about275 Pa·s, about 300 Pa·s, about 400 Pa·s, about 500 Pa·s, about 600Pa·s, about 700 Pa·s, about 750 Pa·s, about 800 Pa·s, about 900 Pa·s,about 1,000 Pa·s, about 1,100 Pa·s, or about 1,200 Pa·s. In otheraspects of this embodiment, a composition disclosed herein exhibits adynamic viscosity of, e.g., at most 10 Pa·s, at most 20 Pa·s, at most 30Pa·s, at most 40 Pa·s, at most 50 Pa·s, at most 60 Pa·s, at most 70Pa·s, at most 80 Pa·s, at most 90 Pa·s, at most 100 Pa·s, at most 125Pa·s, at most 150 Pa·s, at most 175 Pa·s, at most 200 Pa·s, at most 225Pa·s, at most 250 Pa·s, at most 275 Pa·s, at most 300 Pa·s, at most 400Pa·s, at most 500 Pa·s, at most 600 Pa·s, at most 700 Pa·s, at most 750Pa·s, at most 800 Pa·s, at most 900 Pa·s, or at most 1000 Pa·s. In yetother aspects of this embodiment, a composition disclosed hereinexhibits a dynamic viscosity of, e.g., about 10 Pa·s to about 100 Pa·s,about 10 P·as to about 150 Pa·s, about 10 Pa·s to about 250 Pa·s, about50 Pa·s to about 100 Pa·s, about 50 Pa·s to about 150 Pa·s, about 50Pa·s to about 250 Pa·s, about 100 Pa·s to about 500 Pa·s, about 100 Pa·sto about 750 Pa·s, about 100 Pa·s to about 1,000 Pa·s, about 100 Pa·s toabout 1,200 Pa·s, about 300 Pa·s to about 500 Pa·s, about 300 Pa·s toabout 750 Pa·s, about 300 Pa·s to about 1,000 Pa·s, or about 300 Pa·s toabout 1,200 Pa·s.

Aspects of the present specification provide, in part, a compositiondisclosed herein is injectable. As used herein, the term “injectable”refers to a material having the properties necessary to administer thecomposition into a skin region of an individual using an injectiondevice with a fine needle. As used herein, the term “fine needle” refersto a needle that is 27 gauge or smaller. Injectability of a compositiondisclosed herein can be accomplished by sizing the hydrogel particles asdiscussed above.

In aspect of this embodiment, a composition disclosed herein isinjectable through a fine needle. In other aspects of this embodiment, acomposition disclosed herein is injectable through a needle of, e.g.,about 27 gauge, about 30 gauge, or about 32 gauge. In yet other aspectsof this embodiment, a composition disclosed herein is injectable througha needle of, e.g., 27 gauge or smaller, 30 gauge or smaller, or 32 gaugeor smaller. In still other aspects of this embodiment, a compositiondisclosed herein is injectable through a needle of, e.g., about 27 gaugeto about 32 gauge.

In aspects of this embodiment, a composition disclosed herein can beinjected with an extrusion force of about 60 N, about 55 N, about 50 N,about 45 N, about 40 N, about 35 N, about 30 N, about 25 N, about 20 N,or about 15 N. In other aspects of this embodiment, a compositiondisclosed herein can be injected through a 27 gauge needle with anextrusion force of about 60 N or less, about 55 N or less, about 50 N orless, about 45 N or less, about 40 N or less, about 35 N or less, about30 N or less, about 25 N or less, about 20 N or less, about 15 N orless, about 10 N or less, or about 5 N or less. In yet other aspects ofthis embodiment, a composition disclosed herein can be injected througha 30 gauge needle with an extrusion force of about 60 N or less, about55 N or less, about 50 N or less, about 45 N or less, about 40 N orless, about 35 N or less, about 30 N or less, about 25 N or less, about20 N or less, about 15 N or less, about 10 N or less, or about 5 N orless. In still other aspects of this embodiment, a composition disclosedherein can be injected through a 32 gauge needle with an extrusion forceof about 60 N or less, about 55 N or less, about 50 N or less, about 45N or less, about 40 N or less, about 35 N or less, about 30 N or less,about 25 N or less, about 20 N or less, about 15 N or less, about 10 Nor less, or about 5 N or less.

Aspects of the present specification provide, in part, a compositiondisclosed herein exhibits cohesiveness. Cohesion or cohesive attraction,cohesive force, or compression force is a physical property of amaterial, caused by the intermolecular attraction between like-moleculeswithin the material that acts to unite the molecules. A compositionshould be sufficiently cohesive as to remain localized to a site ofadministration. Additionally, in certain applications, a sufficientcohesiveness is important for a composition to retain its shape, andthus functionality, in the event of mechanical load cycling. As such, inone embodiment, a composition exhibits strong cohesive attraction, onpar with water. In another embodiment, a composition exhibits lowcohesive attraction. In yet another embodiment, a composition exhibitssufficient cohesive attraction to remain localized to a site ofadministration. In still another embodiment, a composition exhibitssufficient cohesive attraction to retain its shape. In a furtherembodiment, a composition exhibits sufficient cohesive attraction toretain its shape and functionality.

In aspects of this embodiment, a composition disclosed herein has acompression force of about 10 grams-force, about 20 grams-force, about30 grams-force, about 40 grams-force, about 50 grams-force, about 60grams-force, about 70 grams-force, about 80 grams-force, about 90grams-force, about 100 grams-force, about 200 grams-force, about 300grams-force, about 400 grams-force, about 500 grams-force, about 600grams-force, about 700 grams-force, or about 800 grams-force. In otheraspects of this embodiment, a composition disclosed herein has acompression force of at least 500 grams-force, at least 600 grams-force,at least 700 grams-force, at least 800 grams-force, at least 900grams-force, at least 1000 grams-force, at least 1250 grams-force, atleast 1500 grams-force, at least 1750 grams-force, at least 2000grams-force, at least 2250 grams-force, at least 2500 grams-force, atleast 2750 grams-force, or at least 3000 grams-force. In other aspectsof this embodiment, a composition disclosed herein has a compressionforce of at most 10 grams-force, at most 20 grams-force, at most 30grams-force, at most 40 grams-force, at most 50 grams-force, at most 60grams-force, at most 70 grams-force, at most 80 grams-force, at most 90grams-force, at most 100 grams-force, at most 200 grams-force, at most300 grams-force, at most 400 grams-force, at most 500 grams-force, atmost 600 grams-force, at most 700 grams-force, or at most 800grams-force.

In yet other aspects of this embodiment, a composition disclosed hereinhas a compression force of about 10 grams-force to about 50 grams-force,about 25 grams-force to about 75 grams-force, about 50 grams-force toabout 150 grams-force, about 100 grams-force to about 200 grams-force,about 100 grams-force to about 300 grams-force, about 100 grams-force toabout 400 grams-force, about 100 grams-force to about 500 grams-force,about 200 grams-force to about 300 grams-force, about 200 grams-force toabout 400 grams-force, about 200 grams-force to about 500 grams-force,about 200 grams-force to about 600 grams-force, about 200 grams-force toabout 700 grams-force, about 300 grams-force to about 400 grams-force,about 300 grams-force to about 500 grams-force, about 300 grams-force toabout 600 grams-force, about 300 grams-force to about 700 grams-force,about 300 grams-force to about 800 grams-force, about 400 grams-force toabout 500, about 400 grams-force to about 600, about 400 grams-force toabout 700, about 400 grams-force to about 800, about 500 grams-force toabout 600 grams-force, about 500 grams-force to about 700 grams-force,about 500 grams-force to about 800 grams-force, about 600 grams-force toabout 700 grams-force, about 600 grams-force to about 800 grams-force,about 700 grams-force to about 800 grams-force, about 1000 grams-forceto about 2000 grams-force, about 1000 grams-force to about 3000grams-force, or about 2000 grams-force to about 3000 grams-force.

Aspects of the present hydrogel formulations provide, in part, asurfactant. As used herein, the term “surfactant” refers to a natural orsynthetic amphiphilic compound. A surfactant can be non-ionic,zwitterionic, or ionic. It is envisioned that any surfactant is usefulin making a hydrogel formulation disclosed in the present specification,with the proviso that a therapeutically effective amount of the hydrogelformulation is recovered using this surfactant amount. Non-limitingexamples of surfactants include polysorbates like polysorbate 20 (TWEEN®20), polysorbate 40 (TWEEN® 40), polysorbate 60 (TWEEN® 60), polysorbate61 (TWEEN® 61), polysorbate 65 (TWEEN® 65), polysorbate 80 (TWEEN® 80),and polysorbate 81 (TWEEN® 81); poloxamers (polyethylene-polypropylenecopolymers), like Poloxamer 124 (PLURONIC® L44), Poloxamer 181(PLURONIC® L61), Poloxamer 182 (PLURONIC® L62), Poloxamer 184 (PLURONIC®L64), Poloxamer 188 (PLURONIC® F68), Poloxamer 237 (PLURONIC® F87),Poloxamer 338 (PLURONIC® L108), Poloxamer 407 (PLURONIC® F127),polyoxyethyleneglycol dodecyl ethers, like BRIJ® 30, and BRIJ® 35;2-dodecoxyethanol (LUBROL®-PX); polyoxyethylene octyl phenyl ether(TRITON® X-100); sodium dodecyl sulfate (S DS);3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS);3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate(CHAPSO); sucrose monolaurate; and sodium cholate. Other non-limitingexamples of surfactant excipients can be found in, e.g., PharmaceuticalDosage Forms and Drug Delivery Systems (Howard C. Ansel et al., eds.,Lippincott Williams & Wilkins Publishers, 7^(th) ed. 1999); Remington:The Science and Practice of Pharmacy (Alfonso R. Gennaro ed.,Lippincott, Williams & Wilkins, 20^(th) ed. 2000); Goodman & Gilman'sThe Pharmacological Basis of Therapeutics (Joel G. Hardman et al., eds.,McGraw-Hill Professional, 10^(th) ed. 2001); and Handbook ofPharmaceutical Excipients (Raymond C. Rowe et al., APhA Publications,4^(th) edition 2003), each of which is hereby incorporated by referencein its entirety.

In aspects of this embodiment, a hydrogel formulation comprises apolysorbate, a poloxamer, a polyoxyethyleneglycol dodecyl ether,2-dodecoxyethanol, polyoxyethylene octyl phenyl ether, sodium dodecylsulfate, 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate,3-[(3-Cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate,sucrose monolaurate; or sodium cholate.

Aspects of the present specification provide, in part, a method oftreating a soft tissue condition of an individual by administering acomposition disclosed herein. As used herein, the term “treating,”refers to reducing or eliminating in an individual a cosmetic orclinical symptom of a soft tissue condition characterized by a softtissue imperfection, defect, disease, and/or disorder; or delaying orpreventing in an individual the onset of a cosmetic or clinical symptomof a condition characterized by a soft tissue imperfection, defect,disease, and/or disorder. For example, the term “treating” can meanreducing a symptom of a condition characterized by a soft tissue defect,disease, and/or disorder by, e.g., at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90% or at least 100%. The effectiveness of a compound disclosed hereinin treating a condition characterized by a soft tissue defect, disease,and/or disorder can be determined by observing one or more cosmetic,clinical symptoms, and/or physiological indicators associated with thecondition. An improvement in a soft tissue defect, disease, and/ordisorder also can be indicated by a reduced need for a concurrenttherapy. Those of skill in the art will know the appropriate symptoms orindicators associated with specific soft tissue defect, disease, and/ordisorder and will know how to determine if an individual is a candidatefor treatment with a compound or composition disclosed herein.

A composition or compound is administered to an individual. Anindividual is typically a human being. Typically, any individual who isa candidate for a conventional procedure to treat a soft tissuecondition is a candidate for a method disclosed herein. In addition, thepresently disclosed compositions and methods may apply to individualsseeking a small/moderate enlargement, shape change or contour alterationof a body part or region, which may not be technically possible oraesthetically acceptable with existing soft tissue implant technology.Pre-operative evaluation typically includes routine history and physicalexamination in addition to thorough informed consent disclosing allrelevant risks and benefits of the procedure.

The composition and methods disclosed herein are useful in treating asoft tissue condition. A soft tissue condition includes, withoutlimitation, a soft tissue imperfection, defect, disease, and/ordisorder. Non-limiting examples of a soft tissue condition includebreast imperfection, defect, disease and/or disorder, such as, e.g., abreast augmentation, a breast reconstruction, mastopexy, micromastia,thoracic hypoplasia, Poland's syndrome, defects due to implantcomplications like capsular contraction and/or rupture; a facialimperfection, defect, disease or disorder, such as, e.g., a facialaugmentation, a facial reconstruction, Parry-Romberg syndrome, lupuserythematosus profundus, dermal divots, sunken checks, thin lips, nasalimperfections or defects, retro-orbital imperfections or defects, afacial fold, line and/or wrinkle like a glabellar line, a nasolabialline, a perioral line, and/or a marionette line, and/or other contourdeformities or imperfections of the face; a neck imperfection, defect,disease or disorder; a skin imperfection, defect, disease and/ordisorder; other soft tissue imperfections, defects, diseases and/ordisorders, such as, e.g., an augmentation or a reconstruction of theupper arm, lower arm, hand, shoulder, back, torso including abdomen,buttocks, upper leg, lower leg including calves, foot including plantarfat pad, eye, genitals, or other body part, region or area, or a diseaseor disorder affecting these body parts, regions or areas; urinaryincontinence, fecal incontinence, other forms of incontinence; andgastroesophageal reflux disease (GERD).

The amount of a composition used with any of the methods as disclosedherein will typically be determined based on the alteration and/orimprovement desired, the reduction and/or elimination of a soft tissuecondition symptom desired, the clinical and/or cosmetic effect desiredby the individual and/or physician, and the body part or region beingtreated. The effectiveness of composition administration may bemanifested by one or more of the following clinical and/or cosmeticmeasures: altered and/or improved soft tissue shape, altered and/orimproved soft tissue size, altered and/or improved soft tissue contour,altered and/or improved tissue function, tissue ingrowth support and/ornew collagen deposition, sustained engraftment of composition, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign material.

For example, for breast augmentation procedures, effectiveness of thecompositions and methods may be manifested by one or more of thefollowing clinical and/or cosmetic measures: increased breast size,altered breast shape, altered breast contour, sustained engraftment,reduction in the risk of capsular contraction, decreased rate ofliponecrotic cyst formation, improved patient satisfaction and/orquality of life, and decreased use of breast implant.

As another example, effectiveness of the compositions and methods intreating a facial soft tissue may be manifested by one or more of thefollowing clinical and/or cosmetic measures: increased size, shape,and/or contour of facial feature like increased size, shape, and/orcontour of lip, cheek or eye region; altered size, shape, and/or contourof facial feature like altered size, shape, and/or contour of lip, cheekor eye region shape; reduction or elimination of a wrinkle, fold or linein the skin; resistance to a wrinkle, fold or line in the skin;rehydration of the skin; increased elasticity to the skin; reduction orelimination of skin roughness; increased and/or improved skin tautness;reduction or elimination of stretch lines or marks; increased and/orimproved skin tone, shine, brightness and/or radiance; increased and/orimproved skin color, reduction or elimination of skin paleness;sustained engraftment of composition; decreased side effects; improvedpatient satisfaction and/or quality of life.

As yet another example, for urinary incontinence procedures,effectiveness of the compositions and methods for sphincter support maybe manifested by one or more of the following clinical measures:decreased frequency of incontinence, sustained engraftment, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign filler.

The amount of a composition used with any of the methods disclosedherein will typically be a therapeutically effective amount. As usedherein, the term “therapeutically effective amount” is synonymous with“effective amount”, “therapeutically effective dose”, and/or “ effectivedose” and refers to the amount of compound that will elicit thebiological, cosmetic or clinical response being sought by thepractitioner in an individual in need thereof. As a non-limitingexample, an effective amount is an amount sufficient to achieve one ormore of the clinical and/or cosmetic measures disclosed herein. Theappropriate effective amount to be administered for a particularapplication of the disclosed methods can be determined by those skilledin the art, using the guidance provided herein. For example, aneffective amount can be extrapolated from in vitro and in vivo assays asdescribed in the present specification. One skilled in the art willrecognize that the condition of the individual can be monitoredthroughout the course of therapy and that the effective amount of acomposition disclosed herein that is administered can be adjustedaccordingly.

In aspects of this embodiment, the amount of a composition administeredis, e.g., 0.01 g, 0.05 g, 0.1 g, 0.5 g, 1 g, 5 g, 10 g, 20 g, 30 g, 40g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g, 150 g, or 200 g. In otheraspects of this embodiment, the amount of a composition administered is,e.g., about 0.01 g to about 0.1 g, about 0.1 g to about 1 g, about 1 gto about 10 g, about 10 g to about 100 g, or about 50 g to about 200 g.In yet other aspects of this embodiment, the amount of a compositionadministered is, e.g., 0.01 mL, 0.05 mL, 0.1 mL, 0.5 mL, 1 mL, 5 mL, 10mL, 20 mL, 30 mL, 40 mL, 50 mL, 60 mL, 70 g, 80 mL, 90 mL, 100 mL, 150mL, or 200 mL. In other aspects of this embodiment, the amount of acomposition administered is, e.g., about 0.01 mL to about 0.1 mL, about0.1 mL to about 1 mL, about 1 mL to about 10 mL, about 10 mL to about100 mL, or about 50 mL to about 200 mL.

Aspects of the present invention provide, in part, administering acomposition disclosed herein. As used herein, the term “administering”means any delivery mechanism that provides a composition disclosedherein to an individual that potentially results in a clinically,therapeutically, or experimentally beneficial result. The actualdelivery mechanism used to administer a composition to an individual canbe determined by a person of ordinary skill in the art by taking intoaccount factors, including, without limitation, the type of skincondition, the location of the skin condition, the cause of the skincondition, the severity of the skin condition, the degree of reliefdesired, the duration of relief desired, the particular compositionused, the rate of excretion of the particular composition used, thepharmacodynamics of the particular composition used, the nature of theother compounds included in the particular composition used, theparticular route of administration, the particular characteristics,history and risk factors of the individual, such as, e.g., age, weight,general health and the like, or any combination thereof. In an aspect ofthis embodiment, a composition disclosed herein is administered to askin region of an individual by injection.

The route of administration of composition administered to an individualpatient will typically be determined based on the cosmetic and/orclinical effect desired by the individual and/or physician and the bodypart or region being treated. A composition disclosed herein may beadministered by any means known to persons of ordinary skill in the artincluding, without limitation, syringe with needle, catheter, topically,or by direct surgical implantation. The composition disclosed herein canbe administered into a skin region such as, e.g., a dermal region or ahypodermal region. In addition, a composition disclosed herein can beadministered once, or over a plurality of times. Ultimately, the timingused will follow quality care standards.

For a breast soft tissue replacement procedure, the route ofadministration may include axillary, periareolar, and/or inframammaryroutes. Alternatively or in addition, a composition may be deliveredthrough a transaxillary endoscopic subpectoral approach. For a facialsoft tissue replacement procedure, the route of administration can befrontal, temporal, zygomatic, periocular, amdibula, perioral or chinroutes. In urinary incontinence procedures, the route of administrationmay include transurethral or periurethral routes. Alternatively or inaddition, administration may be delivered via an antegrade route. Theroutes discussed herein do not exclude the use of multiple routes toachieve the desired clinical effect.

Aspects of the present invention provide, in part, a dermal region. Asused herein, the term “dermal region” refers to the region of skincomprising the epidermal-dermal junction and the dermis including thesuperficial dermis (papillary region) and the deep dermis (reticularregion). The skin is composed of three primary layers: the epidermis,which provides waterproofing and serves as a barrier to infection; thedermis, which serves as a location for the appendages of skin; and thehypodermis (subcutaneous adipose layer). The epidermis contains no bloodvessels, and is nourished by diffusion from the dermis. The main type ofcells which make up the epidermis are keratinocytes, melanocytes,Langerhans cells and Merkels cells.

The dermis is the layer of skin beneath the epidermis that consists ofconnective tissue and cushions the body from stress and strain. Thedermis is tightly connected to the epidermis by a basement membrane. Italso harbors many Mechanoreceptor/nerve endings that provide the senseof touch and heat. It contains the hair follicles, sweat glands,sebaceous glands, apocrine glands, lymphatic vessels and blood vessels.The blood vessels in the dermis provide nourishment and waste removalfrom its own cells as well as from the Stratum basale of the epidermis.The dermis is structurally divided into two areas: a superficial areaadjacent to the epidermis, called the papillary region, and a deepthicker area known as the reticular region.

The papillary region is composed of loose areolar connective tissue. Itis named for its fingerlike projections called papillae that extendtoward the epidermis. The papillae provide the dermis with a “bumpy”surface that interdigitates with the epidermis, strengthening theconnection between the two layers of skin. The reticular region liesdeep in the papillary region and is usually much thicker. It is composedof dense irregular connective tissue, and receives its name from thedense concentration of collagenous, elastic, and reticular fibers thatweave throughout it. These protein fibers give the dermis its propertiesof strength, extensibility, and elasticity. Also located within thereticular region are the roots of the hair, sebaceous glands, sweatglands, receptors, nails, and blood vessels. Tattoo ink is held in thedermis. Stretch marks from pregnancy are also located in the dermis.

The hypodermis lies below the dermis. Its purpose is to attach thedermal region of the skin to underlying bone and muscle as well assupplying it with blood vessels and nerves. It consists of looseconnective tissue and elastin. The main cell types are fibroblasts,macrophages and adipocytes (the hypodermis contains 50% of body fat).Fat serves as padding and insulation for the body.

In an aspect of this embodiment, a composition disclosed herein isadministered to a skin region of an individual by injection into adermal region or a hypodermal region. In aspects of this embodiment, acomposition disclosed herein is administered to a dermal region of anindividual by injection into, e.g., an epidermal-dermal junction region,a papillary region, a reticular region, or any combination thereof.

Aspects of the present specification disclose, in part, a method oftreating a soft tissue condition of an individual, the method comprisingthe steps of administering a composition disclosed herein to a site ofthe soft tissue condition of the individual, wherein the administrationof the composition improves the soft tissue condition, thereby treatingthe soft tissue condition. In aspects of this embodiment, a soft tissuecondition is a breast tissue condition, a facial tissue condition, aneck condition, a skin condition, an upper arm condition, a lower armcondition, a hand condition, a shoulder condition, a back condition, atorso including abdominal condition, a buttock condition, an upper legcondition, a lower leg condition including calf condition, a footcondition including plantar fat pad condition, an eye condition, agenital condition, or a condition effecting another body part, region orarea.

Other aspects of the present specification disclose, in part, a methodof treating a skin condition comprises the step of administering to anindividual suffering from a skin condition a composition disclosedherein, wherein the administration of the composition improves the skincondition, thereby treating the skin condition. In an aspect of thisembodiment, a skin condition is a method of treating skin dehydrationcomprises the step of administering to an individual suffering from skindehydration a composition disclosed herein, wherein the administrationof the composition rehydrates the skin, thereby treating skindehydration. In another aspect of this embodiment, a method of treatinga lack of skin elasticity comprises the step of administering to anindividual suffering from a lack of skin elasticity a compositiondisclosed herein, wherein the administration of the compositionincreases the elasticity of the skin, thereby treating a lack of skinelasticity. In yet another aspect of this embodiment, a method oftreating skin roughness comprises the step of administering to anindividual suffering from skin roughness a composition disclosed herein,wherein the administration of the composition decreases skin roughness,thereby treating skin roughness. In still another aspect of thisembodiment, a method of treating a lack of skin tautness comprises thestep of administering to an individual suffering from a lack of skintautness a composition disclosed herein, wherein the administration ofthe composition makes the skin tauter, thereby treating a lack of skintautness.

In a further aspect of this embodiment, a method of treating a skinstretch line or mark comprises the step of administering to anindividual suffering from a skin stretch line or mark a compositiondisclosed herein, wherein the administration of the composition reducesor eliminates the skin stretch line or mark, thereby treating a skinstretch line or mark. In another aspect of this embodiment, a method oftreating skin paleness comprises the step of administering to anindividual suffering from skin paleness a composition disclosed herein,wherein the administration of the composition increases skin tone orradiance, thereby treating skin paleness. In another aspect of thisembodiment, a method of treating skin wrinkles comprises the step ofadministering to an individual suffering from skin wrinkles acomposition disclosed herein, wherein the administration of thecomposition reduces or eliminates skin wrinkles, thereby treating skinwrinkles. In yet another aspect of this embodiment, a method of treatingskin wrinkles comprises the step of administering to an individual acomposition disclosed herein, wherein the administration of thecomposition makes the skin resistant to skin wrinkles, thereby treatingskin wrinkles.

Aspects of the present specification provide, in part, administration ofa composition disclosed herein wherein such administration promotes newcollagen deposition. The compositions comprising a silk fibroin hydrogelcomponent or particle and matrix polymer hydrogel component or particlesupport tissue ingrowth and new deposition of collagen (Example 21).

In an embodiment, administration of a composition comprising a silkfibroin hydrogel component and a matrix polymer hydrogel component asdisclosed herein increases new collagen deposition. In aspects of thisembodiment, administration of a composition comprising a silk fibroinhydrogel component and a matrix polymer hydrogel component as disclosedherein increases new collagen deposition by about 10%, about 20%, about30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%,or about 100%, relative to a the same or similar composition comprisingthe matrix polymer hydrogel component, but lacking the silk fibroinhydrogel component. In other aspects of this embodiment, administrationof a composition comprising a silk fibroin hydrogel component and amatrix polymer hydrogel component as disclosed herein increases newcollagen deposition by at least 25%, at least 50%, at least 75%, atleast 100%, at least 125%, at least 150%, at least 175%, at least 200%,at least 225%, at least 250%, at least 275%, or at least 300%, relativeto a the same or similar composition comprising the matrix polymerhydrogel component, but lacking the silk fibroin hydrogel component. Inyet other aspects of this embodiment, administration of a compositioncomprising a silk fibroin hydrogel component and a matrix polymerhydrogel component as disclosed herein increases new collagen depositionby about 10% to about 100%, about 50% to about 150%, about 100% to about200%, about 150% to about 250%, about 200% to about 300%, about 350% toabout 450%, about 400% to about 500%, about 550% to about 650%, about600% to about 700%, relative to a the same or similar compositioncomprising the matrix polymer hydrogel component, but lacking the silkfibroin hydrogel component.

EXAMPLES

The following examples illustrate representative embodiments nowcontemplated, but should not be construed to limit the disclosedpurified silk fibroin and method for purifying such silk fibroins,hydrogels comprising such silk fibroin with or without an amphiphilicpeptide and methods for making hydrogels comprising such silk fibroinand the use of silk fibroin hydrogels in a variety of medical uses,including, without limitation fillers for tissue space, templates fortissue reconstruction or regeneration, scaffolds for cells in tissueengineering applications and for disease models, a surface coating toimprove medical device function, or as a platform for drug delivery.

Example 1 Silk Sericin Extraction

Silk fibroin for generation of the hydrogel was obtained in the form ofdegummed B. mori silk at a size of 20 denier −22 denier (38 μm±5.6 μmdiameter). This degummed silk was further processed in order to removethe inherently present and potentially antigenic protein glue, sericinthat conjoins independent fibroin filaments. This was done as describedpreviously herein. Following removal of sericin, the pure fibroin wasdried carefully to ambient humidity levels using a laminar flow hood.

Example 2 Generation of Silk Fibroin Solution

Silk fibroin filaments, cleaned of their sericin and rinsed free ofinsoluble debris and ionic contaminants were used for the generation ofan aqueous silk solution. These silk fibers were added to a solution of9.3M LiBr and purified water (e.g., MILLI-Q® Ultrapure WaterPurification Systems) (Millipore, Billerica, Mass.) to make a solutionconsisting of 20% pure silk (% w/v). This mixture was then heated to atemperature of 60° C. and digested for a period of four hours. A totalof 12 mL of the resultant solution was then loaded into a 3 mL-12 mLSlide-A-Lyzer dialysis cassette (Pierce Biotechnology, Inc., Rockford,Ill.) (molecular weight cutoff of 3.5 kD) and placed into a beakercontaining purified water as a dialysis buffer at a volume of 1 L waterper 12 mL cassette of silk solution. The beakers were placed on stirplates and stirred continuously for the duration of the dialysis.Changes of dialysis buffer occurred at 1, 4, 12, 24, and 36 hours ofprocessing time.

Following dialysis, the solution was removed from the cassettes by meansof a syringe and needle and centrifuged at 30,000 g relative centrifugalforce (RCF) at 4° C. for 30 minutes, decanting the supernatant (silksolution) into a clean centrifuge tube, then repeating thecentrifugation for a further 30 minutes. This process of centrifugationis beneficial for removal of insoluble particulate debris associatedwith the silk solution both prior to and following after dialysis. It isbelieved that such insoluble debris could serve as antigens in vivo orperhaps nucleation points about which gelation of the silk could occur,shortening storage life of the solution and compromising the uniformityof the gelation system. After completion of the second centrifugation,the supernatant was again collected and stored at 4° C. until needed. Toconfirm uniformity of the dialysis product, known volumes of thesolution were collected, massed, and then dried completely throughlyophilization. These lyophilized samples were then massed and the drymass of solution compared to initial solution volume to determinepercent silk present per unit volume of solution. Additionally, thesolution was assessed via X-ray Photoelectron Spectroscopy (XPS)analysis to ensure that no detectable quantities of Li⁺ or Br⁻ ions werepresent in the solution.

Example 3 Induction of Gelation

A variety of different methods were employed in the course of hydrogeldevelopment for the purposes of contrasting and comparing certainrelevant properties of various formulae. Regardless of the nature inwhich the gelation process was carried out, the final determination thata “gel” state had been reached was applied uniformly to all groups. Asolution or composite of solutions (i.e., silk solution blended with anenhancer or enhancer solution) was considered a gel after observingformation of a uniform solid phase throughout the entire volume,generally opaque and white in appearance.

Samples to be produced by passive gelation were not exposed to anyenhancer additives. These gels were produced by measuring a volume ofsilk solution into a casting vessel, for the purposes of theseexperiments, polypropylene tubes sealed against air penetration andwater loss, and the sample allowed to stand under ambient roomconditions (nominally 20-24° C., 1 atm, 40% relative humidity) untilfully gelled. Care was taken to ensure uniformity of casting vesselsmaterial of construction across groups so as to avoid potentialinfluence from surface effects. These effects may serve to enhance orinhibit gelation and may be caused by factors including but not limitedto siliconization, surface roughness, surface charge, debriscontamination, surface hydrophobicity/hydrophilicity, and altered masstransfer dynamics.

Samples produced by means of a 23RGD-induced process were made in one oftwo ways, the first being direct addition of 23RGD in a pre-determinedratio to the silk solution without any sort of reconstitution. The 23RGD(obtained as a desiccated fine powder form) was blended into a measuredvolume of 8% silk solution within the casting vessel by pipetting usinga 1000 μL pipette. These gels were then cast in polypropylene tubes,sealed against air penetration and water loss, and the sample wasallowed to stand under ambient room conditions (nominally 20-24° C., 1atm, 40% relative humidity) until fully gelled.

The 23RGD-induced gels were also produced by first dissolving the 23RGDpowder in purified water. The concentration of this solution wasdetermined based upon the amount of 23RGD to be introduced into a geland the final concentration of silk desired in the gel. In the case of4% silk gels enhanced with 23RGD, quantities of water equal to theamount of 8% silk solution to be used in the gel were used for thedissolution of appropriate quantities of 23RGD. In the case of gelsinduced by addition of 23RGD to be generated at a molar ratio of 3:123RGD:silk, a quantity of 23RGD was dissolved in 1 mL of water per 1 mLof 8% silk solution to be gelled. This mixing was performed in thecasting vessel as well, being accomplished by means of rapid pipettingwith a 1000 μL pipette when appropriate. These gels were then cast inpolypropylene tubes, sealed against air penetration and water loss, andthe sample was allowed to stand under ambient room conditions (nominally20-24° C., 1 atm, 40% relative humidity) until fully gelled.

Samples produced by means of ethanol-enhanced gelation (EEG) weregenerated by means of directly adding ethanol to a measured volume of 8%silk solution in the casting vessel. The ethanol is added in a quantitysuch that the volume added should yield a volumetric dilution of the 8%silk solution resulting in the final required concentration of silkwithin the gel, assuming minimal volume loss due to miscibility of theorganic added. The mixture of ethanol and silk solution is then mixed bymeans of pipetting with a 1000 μL pipette when appropriate. These gelswere then cast in polypropylene tubes, sealed against air penetrationand water loss, and the sample was allowed to stand under ambient roomconditions (nominally 20-24° C., 1 atm, 40% relative humidity) untilfully gelled.

Samples produced by a combined 23RGD-ethanol effect (RGDEEG) weregenerated using a solution of 90% ethanol, 10% purified water andappropriate quantities of 23RGD dissolved in this solvent. It was notpossible to readily dissolve 23RGD in pure ethanol and it was believedthat undissolved 23RGD might cause poor distribution of the peptidethroughout the gel phase. As a result, it was determined that since asolution of ethanol and water offering similar gelation accelerationcharacteristics to a pure ethanol solution and reasonable 23RGDsolubility would be an acceptable alternative. A solution of 90% ethanoland 10% water met both of these criteria and as a result was used forgeneration of these gels. The 23RGD concentration of this ethanolsolution was determined based upon the amount of 23RGD to be introducedinto a gel and the final concentration of silk desired in the gel. Inthe case of 4% silk gels enhanced with 23RGD, quantities of 90% ethanolequal to the amount of 8% silk solution to be used in the gel were usedfor the dissolution of appropriate quantities of 23RGD. In the case ofgels induced by addition of 23RGD to be generated at a molar ratio of3:1 23RGD:silk, a quantity of 23RGD was dissolved in 1 mL of 90& ethanolper 1 mL of 8% silk solution to be gelled. This mixing was performed inthe casting vessel as well, being accomplished by means of rapidpipetting with a 1000 μL pipette when appropriate. These gels were thencast in polypropylene tubes, sealed against air penetration and waterloss, and the sample was allowed to stand under ambient room conditions(nominally 20-24° C., 1 atm, 40% relative humidity) until fully gelled.

Silk gelation times were determined by casting gels according to themethods above, the exception being that gels were mixed not throughpipetting, but through vigorous mechanical shaking. These studies wereconducted using 1.5 mL microcentrifuge tubes as casting vessels withsample groups of N=6 used for each gel formulation (FIG. 1). Thedetermination that a “gel” state had been reached was made in the methodas described above, based upon observation of a uniform solid phasethroughout the entire volume, generally opaque and white in appearance.

Gelation time varied widely depending on specific formulation. The 8Psilk samples took 21 days until gelation while the 4P samples required31±1 day (data not shown). EEG samples gelled significantly faster thanPG samples with a 4E sample requiring 27±5.4 seconds for gelation(p≦0.05). EEG samples gelled more rapidly as the concentration ofethanol added increased with time required gelation times of 1770±600 s,670.3±101.0 s, 29.8±5.2 s, 9.7±2.0 s, and 4.2±0.8 s for 6.4E, 6E, 4.8E,4E, and 3.2E respectively. There were significant differences betweenall times except 4.8E and 4E, 4E and 3.2E, and 4.8E and 3.2E. RGDEEGgels generated a tightly localized white fibrous precipitateinstantaneously upon addition of the ethanol solution to the silk andgelled more quickly than PG samples, though they were slower than EEGgels. 4RL, 4RM and 4RH samples took 22.7±2.5 seconds, 38.8±4.5 seconds,and 154.5±5 seconds to gel with 4RH differing significantly from theother RGDEEG formulations.

Gelation timing experiments revealed the time constraints posed by thePG method. Results indicated that, while increased silk concentrationdecreased gelation time, the total time to gel was decreased only from31 days for 4P to 21 days for 8P. This may result from the increasedfrequency of collisions between silk molecules in solution and resultantgel network assembly. Using ethanol directly added to silk solution asan accelerant proved to dramatically decrease the gelation time of thesilk by increasing the volume of ethanol added in a fashion well-modeledby a power function. This increasingly rapid gelation is likely causedby greater competition for hydrating water molecules between silk andethanol coupled with altered electronegativity of the solution, bothfavoring forced aggregation of the silk molecules. Studies conducted onRGDEEG samples revealed that addition of greater concentrations of RGDled to increasing gelation times modeled by an exponential function.This appears counter-intuitive as it was expected that RGD shouldfunction in some capacity to accelerate gelation.

The slowing of gelation in RGDEEG samples may result from difficultiesin silk molecular binding to the RGD-coated silk precipitates, perhapsdue to stearic interference with hydrophobic regions of silk chains.Upon RGD-ethanol accelerant addition to the silk solution, a largequantity of silk-RGD complexes was precipitated from the solution. Itwas noted during the gelation of RGDEEG samples that a fibrillar, white,opaque precipitate was consistently formed within the solution mixtureimmediately upon mixing. This precipitation from solution may beevidence of this rapid assembly of high concentration silk-RGDprecipitates. This formation may be caused by association between silkmicelles and peptide molecules in solution, disruption of the silkmicelles, and rapid assembly of them into a tightly-localized fibrillarstructure. This rapid assembly may progress until driving gradientsgenerated by the differing solvent chemistries provided by the ethanoland water reach an equilibrium state. At this point, silk molecules areable to remain stably in solution with further silk network assemblyoccurring only by slow lengthening of the initially formed precipitates.While this precipitation provided a high number of nucleation points toinitiate completion of a gel network, these nucleation points may be oflimited utility based upon availability of binding sites. The remainingsilk molecules were much slower to assemble as a result. Theseprecipitates also tended to initiate assembly of a peripheral networkcomprised largely of loose α-helix and random coil motifs, possibly dueto interference in silk packing due to the interference of theseparticles.

The hydrogels produced by the methods described above derive substantialbenefit from the ability to more precisely control the time course forits gelation in comparison to that of a conventionally designed and castgel. It is evident from monitoring the time between casting and gelationof the device and similarly cast, non-enhanced or exclusively ethanolmodified gels that 23RGD under certain circumstances may be manipulatedto have an additional accelerant effect upon the process of gelation.This observed enhancer effect both mitigates the time constraints andcontrollability associated with non-modified gels and additionallyalters the manner in which the protein aggregate network is formedrelative to solely ethanol enhanced gels

Example 4 Determination of Residual Ethanol by Colorimetric Analysis

Following gelation of a sample produced with either an ethanol or 23RGDcomponent, the gel was removed from the casting vessel and immersed in abulk of purified water as a rinse buffer. This bulk comprised a volumesuch that the volumetric ratio of water to gel was ≧100:1. The gel waspermitted to lay static in the rinse buffer for a period of 72 hours,changing the water every 12 hours.

Samples of silk gel were evaluated to determine the total residualcontent of ethanol in a series of 23RGD-ethanol- and ethanol-enhancedgels. Briefly, samples of gel (N=4 of each type) generated as describedabove were processed and analyzed using an Ethanol Assay Kit (kit#K620-100 from BioVision Research Prods, Mountain View, Calif.). Samplesof gel were cut to a size of approximately 0.3 cm in height by 0.5 cm indiameter (approximately 250 mg). These samples were massed to thenearest 0.1 mg using an APX-60 (Denver Instrument, Denver Colo.) balanceas per the manufacturer's instructions. These gel samples wereindividually ground using a metal spatula and placed into 250 μL ofMilli-Q water in microcentrifuge tubes. These gels were incubated at 37°C. for a period of 24 hours. After incubation, the gels were centrifugedon an Eppendorf 5415 microcentrifuge with an HA 45-18-11 rotor (Hamburg,Germany) at 18,000 rpm for 30 minutes. At the conclusion of thiscentrifugation step, the supernatant was used as the sample of interestaccording to the instructions provided by the kit manufacturer.Colorimetric analyses of the sample was performed at an absorbance of570 nm using a spectrophotometer, and in conjunction with a standardcurve, residual percentages of ethanol in the gel were calculated (Table1, FIG. 2). It was shown in this process that the leeching step iscapable of substantially removing residual ethanol from the silk gels,as none of these materials exhibited a residual ethanol component ofgreater than 5% ethanol by mass.

TABLE 1 Determination of Residual Ethanol by Colorimetric Analysis SilkEnhancer Enhancer Initial Ethanol Final Ethanol ConcentrationConcentration Solvent Solute Concentration Mean Stdev 2% 90% None 68%2.49% 0.06%  3:1 23RDG:Silk 4.44% 0.13% 10:1 23RDG:Silk 4.77% 0.29% 4%None 45% 2.55% 0.07%  3:1 23RDG:Silk 2.86% 0.08% 10:1 23RDG:Silk 2.97%0.07% 6% None 22.5%  3.12% 0.05%  3:1 23RDG:Silk 3.16% 0.04% 10:123RDG:Silk 2.99% 0.10%

Example 5 23RGD Quantification by HPLC

23RGD-infused gels were studied to quantify the amount of 23RGD bound tothe silk-hydrogel device as well as the quantity of free 23RGD whichmight be rinsed free of the device under relevant conditions. Briefly,samples of 23RGD-infused gel were cast and rinsed according to themethods above, with samples of rinse buffer being collected from eachrinse for subsequent analysis by HPLC. Additionally, subsequent to thelast rinse, the gel samples were mechanically pulverized by means of astainless steel stirring rod and the adsorbed 23RGD removed byincubation for 4 hours in a dissolving buffer. This mixture of gel andsolvent was then centrifuged on an Eppendorf 5415C at 16,000 g RCF for30 minutes. The supernatant was collected and centrifuged another 30minutes at 16,000 g RCF after which time the supernatant was collectedin a sample vial for HPLC analysis. Samples of rinse buffer from thefirst and last rinse were centrifuged in the same fashion after beingdiluted with the same solvent the gel was extracted with in a volumetricratio of 1 part rinse buffer to 4 parts solvent. To ensure23RGD-hydrogel device rinse-exposed surface area was not a limitingfactor, the same rinse and extraction process was performed upon devicespulverized after gelation and before rinsing. The peak area consistentwith 23RGD for each HPLC sample was taken and these data comparedagainst a standard curve generated for 23RGD on the same HPLC unit underidentical handling and run conditions.

The resultant data indicated levels of signal from 23RGD in samplescollected from rinse buffer were just slightly higher than values for23RGD solvent alone and were immeasurable by the standard curve,expected to resolve a relative 23RGD:silk ratio of 0.05:1. Bycomparison, the assay was able to detect a ratio of 3.35:1 in the finalrinsed and extracted 23RGD-enhanced gel.

HPLC data confirmed complete retention of RGD on the silk hydrogelmaterial after the rinse process. This provides not only a functionalRGD component to this specific series of hydrogel formulations, butindication for use of amphiphilic peptides as candidates forintroduction of other components into silk gels. This knowledge might beapplied to a number of other biologically active peptide sequences,though additional work must be done to understand how these specificpeptides might influence gelation and how gelation in turn impacts thefunctionality of these peptides.

Example 6 Silk Gel Dry Massing

Silk gel samples of various 23RGD-ethanol- and ethanol-enhancedformulations were cut into sample cylinders (N=4 of each type) ofapproximately 0.7 cm in height by 0.5 cm in diameter (approximately 500mg). These samples were massed to the nearest 0.1 mg using an APX-60(Denver Instrument, Denver Colo.) balance as per the manufacturer'sinstructions and placed into massed microcentrifuge tubes. After this,the samples were frozen to −80° C. for 24 hours. At the conclusion ofthis time, the samples were placed into a lyophilizer unit for a periodof 96 hours to remove all water content. Following the completion ofthis 96 hour drying, the remaining protein components of the silk gelsamples were massed again and the mass fraction of water in the samplesdetermined.

Gel dry massing showed an increasing percentage of dry mass as RGDcomponent increased in each silk concentration group (FIG. 3). The drymass of 2E was significantly less than 2RL and 2RM (p≦0.05) at1.63±0.30%, 3.85±1.23% and 4.03±0.53% respectively (FIG. 3A). The drymasses of 4E, 4RL and 4RM all differed significantly from each other at4.05±0.10%, 4.56±0.12%, and 5.19±0.18% respectively (FIG. 3B). The drymass of 6E was significantly less than both 6RL and 6RM at 5.84±0.15%,6.53±0.28%, and 6.95±0.40% respectively (FIG. 3C).

The gels, regardless of the silk concentration, showed a statisticallysignificant trend toward decreasing percentage of water mass in each gelmaterial as 23RGD component increased as determined by analysis of eachsilk concentration group with ANOVA (FIG. 4, Tukey post hoc, p<0.05).This phenomenon might be explained by the possibility that the 23RGDcauses formation of a different secondary structure within the silkhydrogels and that this structure might be less hydrophilic thannon-23RGD-enhanced material. It is possible that this may be manifestedin a different ratio of β-sheet structure, α-helix structure, andunordered random coil for 23RGD-treated materials than theircounterparts, tending to favor the more hydrophobic β-sheetconformation.

Silk gel dry mass data revealed that increasing concentrations of bothsilk and RGD in the silk gels increased the percentage of dry mass inthese materials, though the increase from ROD was too large to attributesolely to additional peptide mass. This phenomenon might be explained bythe hypothesized structure of the RGDEEG gels mentioned previouslyrelative to PG and EEG gels. It is likely that the large regions ofpoorly-associated 13-sheet structure in the RGDEEG gels do a poor job atintegrating water into the structure. The inter-connecting regions ofα-helix structures and unordered random coil are able to entrain water,but do so with less success than in the case of the more homogenous EEGgels. It may also be possible that the hydrophilic ROD sequenceinterfered with the dry massing procedure, causing rapid gain of watermass upon exposure of the samples to atmospheric conditions.

Example 7 Enzymatic Bioresorption

Gels specified were subjected to in vitro digestion by a solutionconsisting of non-specific protease mixture. Briefly, gel samples werecast to generate uniform, cylindrical samples of approximately 1 gramtotal weight (about 1 mL of gel). These samples were digested with aprotease obtained from the bacteria Streptomyces griseus (Sigma catalogNo. P-5147) suspended in phosphate buffered saline at a concentration of1 mg/ml. A ratio of 3 mL of protease solution per 1 ml of initial gelwas used for the purposes of this study. The protease solution was addedto a sealed tube containing the gel and incubated for 24 hours at 37° C.with no mechanical mixing. After 24 hours, the solution was drainedthrough a piece of 316 stainless steel woven wire cloth. This permittedretention of all gel particles greater than 50 μm in diameter (gap sizewas 43 μm by 43 μm), those smaller than that were considered to be“bioresorbed” for the purposes of this assay. After thorough draining ofthe solution, the mass of the gel was measured wet, but devoid of excessentrained moisture. The protease solution was then replaced and thesample incubated a further 24 hours at 37° C. This process was repeateduntil the samples were bioresorbed for a total of four days, changingsolutions and massing each day.

PG samples and EEG samples bioresorbed similarly, differingsignificantly only at D4 where 4P samples retained 62.89±4.26% of theoriginal mass and 4E samples retained 53.27±5.45% (p≦0.05) (FIG. 5A). 6Egels incubated in PBS showed no significant mass loss over the course ofthe 4 day incubation (FIG. 5B). EEG silk gels with high concentrationsof fibroin exhibited higher mass retention than lower concentrations atall days. At Day 1 there were significant differences between 2E and allother gel types with 2E, 4E and 6E gels retaining 57.04±10.03%,93.21±9.47%, and 103.98±3.65%, respectively while 6E in PBS retained101.18%±12.01%. At Day 2, there were significant differences againbetween 2E and all other gel types with 2E, 4E and 6E gels retaining36.59±7.07%, 90.60±9.24%, and 103.24±6.38% of the original mass while 6Ein PBS retained 98.28%±12.38%. At Day 3 there were significantdifferences between all gel types in protease, with 2E, 4E and 6E gelsretaining 32.36±10.48%, 67.85±8.82%, and 95.51±8.97% of the originalmass. 6E samples incubated in PBS did not differ from those incubated inprotease, retaining 100.39%±12.73% of the original mass. At Day 4 therewere significant differences between all gel types with 2E, 4E, and 6Egels retaining 28.14±4.75%, 53.27±5.45%, and 81.76%±3.35% of theoriginal mass while 6E in PBS retained 102.45±12.50%. Addition of RGD tosilk gels appeared to slightly decrease the mass retention of thesematerials when subjected to proteolytic bioresorption (FIG. 5C). 4Esamples retained significantly more mass than 4RM and 4RH at Day 2 asthey retained 90.6±9.24%, 74.47±4.55%, and 71.23±6.06% of the initialmasses respectively. There were no further significant differences in 4Esamples relative to 4RM and 4RH samples over the course of thebioresorption assay.

Gel samples treated with 23RGD exhibit a trend toward more rapidbioresorption within the constraints of this particular assay. This wasillustrated at the 4% silk concentration (FIG. 6) and then confirmed ata concentration of 6% silk fibroin in the gel materials (FIG. 7).Significant differences in the bioresorption rates of 23RGD enhancedsamples recorded by two-way ANOVA using a Bonferroni post test (p<0.05),particularly with 6% silk, reinforced the trend. The unique behaviorattributed to 23RGD-enhanced materials may be due in part to its uniqueprotein structure, as the bioresorption method considers particles belowa size of 50 μm to be bioresorbed, regardless of their stability. It maybe possible for a rich beta sheet structure to exist within 23RGD gelswhich is broken up into small, discrete regions by interfering regionsof α-helix structure and random coil which bioresorb more quickly,creating a plethora of tiny, non-resorbed fragments in solution.

In vitro bioresorption of 4P and 4E samples showed both materials had asimilar resistance to proteolysis (FIG. 5A). This is indicative that thebasic process of ethanol-enhanced gelation is capable of generating agel structure rapidly without sacrificing important material properties.It was also shown that increasing the concentration of silk in EEG gelsfrom 2% to 4% to 6% in 2E, 4E, and 6E respectively, substantiallydecreased sample bioresorption mass loss (FIG. 5B). This may correlateto a more homogeneous, stable and resilient gel structure, or simply toa greater quantity of silk molecules to be cleaved by the proteases inorder to bioresorb the samples. In either case, these data clearlyindicate a potential for tailoring of bioresorption time scale of a silkgel material through alteration of the silk protein content of gels. Itwas also illustrated that a 4 day exposure to PBS did not appreciablyalter the mass of 6E samples, providing a preliminary indication thatEEG samples are not substantially degraded by hydrolysis. This is afurther reinforcement of the stability and bulk integrity of these silkgels as many gel materials suffer from limited resilience in vivo due tohigh susceptibility to hydrolysis. Addition of increasing quantities ofRGD to silk gels was shown to slightly increase the rates ofbioresorption mass loss in comparing 4E, 4RM and 4RH (FIG. 5C). Thisbehavior indicates that there may be some structural differences betweenRGDEEG and EEG gels which cause less mass loss in EEG gels as comparedto RGDEEG in this bioresorption assay. This may relate directly to thepreviously proposed idea that RGDEEG materials consist of many smallregions of robust β-sheet structure loosely bound together by a weakinter-connecting matrix of α-helix and unordered random coil structures.This stands in contrast to EEG materials, which are thought to assemblefrom similar, though less prominent and numerous, precipitates into amore homogeneous structure than RGDEEG gels as a result. Theinter-connecting matrix of the RGDEEG gels is therefore more susceptibleto rapid bioresorption through this proteolytic assay than that of EEGgels. While β-sheet regions may remain intact in RGDEEG gels, bulkmaterial integrity is lost as the inter-connecting network is resorbedas are the residual β-sheet particles due to the sieving method used asa cutoff for degradation product particle size. This is indicative thatit may be possible to use varying levels of RGD in order to furthermanipulate the structure and bioresorption profile of a silk gel.

Example 8 Fourier-Transform Infrared Spectrum Capture

Silk hydrogels, 23RGD-ethanol-enhanced 4% silk, 3:1 and 10:1, were castas described above and subjected to proteolytic bioresorption asdescribed above. Additionally, non-bioresorbed control samples wereobtained for sake of analysis via FTIR in quantities of 0.5ml each.Using a Bruker Equinox 55 spectrophotometer (Bruker Optics, Inc.,Billerica, Mass.) coupled with a Pike MIRACLE™ germanium crystal (PIKETechnologies, Madison, Wis.), sample absorbance spectra were obtained.Samples were imaged by pressing them upon the crystal via a pressure armuntil single sample scans indicated viable signal from the material thenperforming a 128-scan integration. Resolution was set to 4 cm⁻¹ with a 1cm⁻¹ interval from a range of 4000 cm⁻¹ to 400 cm⁻¹.

Resultant spectra were subjected to analysis via OPUS 4.2 software(Bruker Optics, Inc). A peak-find feature was used to identify peaksbetween 4000 cm⁻¹ and 600 cm⁻¹, with the search criteria being automaticselection of local inflection points of a second-derivative, nine-pointsmoothing function. Program sensitivity was set to 3.5% for all spectrabased upon operator discretion regarding magnitude of peaks identifiedand likely relevance to compound identification and “fingerprinting”.

Each of the samples subjected to FTIR analysis exhibited a spectrum withvery pronounced peaks at the Amide I band (1600-1700 cm⁻¹) (FIG. 8).Additionally, the specific wave numbers of these peaks are consistentbetween the 23RGD-infused silk fibroin hydrogel and other silk gelgroups. All samples exhibit major peaks at ˜1622 cm⁻¹ and a minorpeak/toe region at ˜1700 cm⁻¹, a pattern associated with a high degreeof β-sheet structure within a sample (FIG. 8). There are alsosimilarities across all samples types at the Amide II band with a majorpeak at ˜1514 cm⁻¹.

Use of the EEG process to produce silk gels did not dramatically impactgel secondary structure but did slightly increase the resistance of thegel formulation to proteolytic bioresorption (FIG. 9A). Evaluation ofcharacteristic FTIR spectra of 4P and 4E gels at Day 0 revealed fewdistinguishing characteristics as both formulations exhibited acharacteristic β-sheet peak around 1622 cm-1 and toe region of β-turn at1700 cm-1. Each sample also had additional portions of β-sheet, β-turn,α-helix, and unordered random coil at 1677 cm⁻¹, 1663 cm⁻¹, 1654 cm⁻¹,and 1645 cm⁻¹ respectively with higher relative quantities of α-helixand random coil appearing in 4P than 4E at Day 0. At Day 4, both samplesshowed pronounced decreases in 1677 cm⁻¹ β-sheet, β-turn, α-helix andrandom coil signal, though this 4P exhibited this to a greater extentthan 4E, indicating preferential resorption of these motifs and greaterresistance to this in 4E gels.

Increasing the final silk concentration of EEG gels had little impact oninitial gel secondary structure, though there was a pronounced increasein β-sheet structures at Day 4 with greater silk concentrations (FIG.98). At Day 0, 2E, 4E, and 6E gels all showed strong signal for 1622cm⁻¹ β-sheet and 1700 cm⁻¹ β-turn strong, with 6E having particularlyprominent peaks in these regions. Each sample also had additionalportions of 1677 cm⁻¹ β-sheet, 1663 cm⁻¹ β-turn, α-helix, and unorderedrandom coil. At Day 4 all gels showed decreases in 1677cm-1 β-sheet,1663 cm⁻¹ β-turn, α-helix and random coil peaks relative to 1622 cm⁻¹β-sheet and β-turn peaks with this behavior being more marked in 4E and6E than 2E. The Day 4 6E sample also showed a more stable β-sheetstructure indicated by a peak shift to lower wave number at ˜1620 cm⁻¹.

Pronounced differences in the 23RGD-ethanol-enhanced andethanol-enhanced spectra only became evident after a four-day period ofbioresorption in protease. The day 4 samples exhibited differencesprimarily in the order of magnitude of certain secondary structuremodalities seen through slight differences in FTIR Amide I band shape.At day 4, the 23RGD-ethanol-enhanced samples exhibit higher levels ofβ-turn structure evidenced by far more pronounced and distinct peaks at˜1700 cm⁻¹ while also showing considerably lower levels of α-helixstructure (1654 cm⁻¹) and unordered random coil (1645 cm⁻¹) structures.For example, FTIR spectra from 4E, 4RM and 4RH all show similarstructures featuring 1622 cm⁻¹ β-sheet and 1700 cm⁻¹ β-turn prominentlywith indications of 1677 cm⁻¹ β-sheet, 1663 cm⁻¹ β-turn, α-helix, andunordered random coil secondary structures (FIG. 9C). At Day 4, 4RM and4RH both show a less pronounced 1677 cm⁻¹ β-sheet, 1663 cm-1 β-turn,α-helix, and random coil component than the 4E sample with 4RH alsoshowing a more stable β-sheet structure, indicated by a peak shift tolower wave number at ˜1620 cm⁻¹. Additionally, a peak shift occurred inboth the 10:1 23RGD-ethanol-enhanced and ethanol-enhanced samples in theβ-strand peak at 1622 cm⁻¹, indicative of increased β-sheet stability.Considered as a whole, the collective peak shifts and peak magnitudesobserved in the spectra at day 4 compared to day 0, all gel typesexperienced substantial strengthening of β-sheet component, likely dueto removal of less-stable α-helix and random coil. These effects weremost pronounced in 23RGD-enhanced gel materials, likely due to intrinsicdifferences in the initial organization of the structural network of thegel materials.

FTIR analysis and comparison of PG, EEG and RGDEEG showed strongbehavioral similarities across all gel groups. Each material exhibitedβ-sheet-dominated secondary protein structures, featuring elements ofα-helical and random coil structures and each resorbed in such a fashionthat the quantities of β-sheet-rich structure increased relative toα-helical and random coil structures. The selective bioresorption ofα-helical and random coil structures indicates that they are likelyfavorably degraded by proteolysis relative to β-sheet structures, thusthe bioresorption profile of a gel might be influenced by altering thebalance between β-sheet motifs and the combination of α-helical andrandom coil structures. An evaluation of ethanol as an accelerantrevealed a minimal effect on silk gel structure at Day 0 as both 4P and4E had high β-sheet contents with α-helical and random coil structures(FIG. 9A). At Day 4 though, there was a slightly greater relativeβ-sheet content in 4E than 4P samples. This may be caused by structuraldifferences in 4E and 4P formulations that were imperceptible at Day 0by ATR-FTIR, possibly in the uniformity and homogeneity of the silkgels. It is possible that the same differences hypothesized between EEGand RGDEEG gels derived from their different extents ofprecipitate/nucleation point formation in early-phase gelation causesdifferences between PG and EEG materials as well. As PG samples are notaccelerated, it is likely that very few nucleation points will formquickly and as a result, the gelation process occurs in a very slow buthomogeneous fashion, allowing for an optimal stearic packing of silkmolecules throughout the solution volume. This results in a consistentprotein structure throughout the final gel volume, corresponding to goodbulk material integrity. This would contrast with EEG gels, as thepreviously postulated nucleation phenomenon associated with RGDEEGmaterials likely occurs with EEG materials as well, though in a lessprominent fashion. This results in a non-uniform distribution of highlyorganized regions of β-sheet held together by α-helical and random coilstructures in the EEG materials relative to the PG materials, withα-helical and random coil degraded more rapidly than β-sheet. This is inkeeping with previous studies which have shown that more poorly packedβ-sheet structures and α-helix structures are more susceptible todegradation. Increasing silk concentration in EEG gels from 2E to 4E to6E revealed the most prominent β-sheet structures in 6E at both Day 0and Day 4 while 2E had considerably more α-helix and random coil at bothdays than 2E and 4E (FIG. 9B). This would seem to indicate that diluteconcentrations of silk in the final hydrogel result in a less denselypacked secondary structure, possibly due to stearic freedom within thegel volume relative to 4% and 6% states. This indicates that silkconcentration may be used to manipulate the secondary structure of silkgel to influence bioresorption. A study of the effect of increasing RGDconcentration indicated that while gels were virtually identical at Day0, the α-helix structure and unordered random coil in 4RM and 4RH gelswere less resilient to bioresorption than in 4E as seen at Day 4 (FIG.9C). This might also be explained by inhomogeneities within the 4RM and4RH gels relative to 4E as mentioned previously. This may beparticularly likely in light of the formation of precipitates observedin RGDEEG samples. This data may be indicative that RGD or a similarpeptide could be used to further tailor the nature of the bioresorptionprofile of silk gels.

These results indicate that silk gels produced through PG, EEG, andRGDEEG result from a two-phase assembly process consisting of nucleationand aggregation. Silk gels contain predominantly β-sheet structure whichis more resistant to in vitro bioresorption than α-helix and randomcoil. EEG gels form more quickly than PG, likely due to a more rapidprecipitation and nucleation event mediated by the effects of ethanol onthe solution solvent phase. EEG gels form a non-homogeneous structurelikely consisting of localized, initially-precipitated β-sheet regionsinter-connected by α-helix and random coil assembled subsequently.RGDEEG gels form a non-homogeneous structure likely consisting oflocalized, initially-precipitated β-sheet regions inter-connected byα-helix and random coil assembled subsequently. RGDEEG gels reachcompletion more slowly than EEG gels due to stearic RGD-mediatedinterference encountered in gel assembly following nucleation. RGDEEGgels are less homogeneous than EEG gels due to these difficultiesassociated with late-phase assembly.

Example 9 Injectable Gel Processing

Silk hydrogels were prepared as described above in Examples 1-4. Gelswere then comminuted by grinding the silk gel to a paste using astainless steel spatula. Gel formulations including PBS were massed withan SI-215 balance (Denver Instrument, Denver Colo.) and the correctvolume percentage of PBS (Invitrogen Corporation, Carlsbad, Calif.) wasblended in with the assumption that both the gel and PBS had a densityof 1 g/ml. Silk hydrogels to be used for in vivo assessment weresubjected to vigorous mechanical pulverization by means of a stainlesssteel stir rod. When specified as containing a saline component, gelswere blended with saline at volumetric ratios based upon the originalvolume of gel (i.e., prior to mechanical disruption) followingpulverizing by the stainless steel bar. This addition of phosphatebuffered saline serves to regulate tonicity of the gel as well asimprove injectability. Following this initial pulverizing, the gel wasfurther disrupted by means of repeated injection through a 26-gaugeneedle in order to decrease overall particle size within the gel andimprove injectability characteristics. In some samples, gel was furtherdisrupted by means of repeated injection first through an 18 g needlerepeatedly until the gel flowed readily, and then the material was thencycled in like fashion through a 23 g needle and 26 g needle.

Example 10 In Vivo Investigation of Silk Hydrogel in Rodent Models

Samples of silk gel which had been processed for implantation orinjection in vivo as described in Example 9 were double-bagged withappropriate sterilization bags for gamma irradiation and sterilized byexposure to a dose of 25 kGy of gamma radiation.

In one trial silk hydrogel samples, both 23RGD-enhanced and native wereimplanted subcutaneously in male Lewis rats having an average weight of400 g. This was done according to protocol#86-04 on file with NewEngland Medical Center's Department of Laboratory Animal Medicine (DLAM)and approved by the Institutional Animal Care and Use Committee (IACUC).On the day of surgery, animals were anesthetized via a ketamine/xylazinesolution injected IM in the animals' hind legs. Following administrationof anesthesia, the skin of the rats was shaved closely and swabbed withalcohol, allowed to dry, swabbed with BETADINE® microbicide (PurduePharma, Stamford, Conn.) then draped with sterile towels. In the case ofimplanted devices, two dorsal midline incisions were made directly overthe spine, the first 0.5 cm below the shoulders and the second 2.5 cmabove the pelvic crest, each 1 cm long each. The incisions were expandedinto 1 cm deep pockets using a blunt dissection technique beneath thepanniculus carnosus at each side yielding 4 potential implant sites.Implants, 3 per animal; each 1 cm×1 cm×0.3 cm in size were inserted intothe pockets without fixation with the final site undergoing the samedissection but replacing the implant with 0.5 mL of sterile salinesolution. The skin was closed with interrupted absorbable sutures.Depending on study, samples were harvested at 7 days, 14 days, 28 days,and/or 57 days after implantation surgery. Gross observations werecollected semi-weekly regarding implant site appearance. After sampleharvest, gross observations of the implants were conducted and sampleswere processed for histological evaluation. Analysis of histology slideswas provided by a trained veterinary pathologist.

Sections were scored for presence (0=none, 1=present) of implantmineralization, cyst formation, fibrosis, sebaceous cell hyperplasia,and focal follicular atrophy. Additionally, the density of inflammatoryresponse (0=none . . . 5=extensive) and extent epidermal hyperplasia(0=none . . . 3=extensive) were graded. These data were reported aspercentages of the highest score possible for the group of slides.Sections were also examined for presence of any particularcharacteristic cell types including lymphocytes, neutrophils,eosinophils, mononuclear giant cells, macrophages, and fibroblasts.Additional commentary relevant to the host response was included at thediscretion of the reviewing pathologist. Prism 4.03 (GraphPad SoftwareInc., San Diego, Calif.) was used to perform analysis of variance(ANOVA) with a significance threshold set at p≦0.05. One-way ANOVA wasused to compare differences average extrusion forces for comminutedgels. For all tests, Tukey's post-hoc test was also performed formultiple comparisons.

Table 2 lists the formulations of silk gel, both 23RGD-ethanol-enhancedand ethanol-enhanced developed and assessed intradermally in a ratmodel. Silk gels explanted from rats at Day 7 were visibly well-definedand easily identifiable with no gross indications of edema, erythema, ortransdermal elimination of material. It was not possible todifferentiate sites of PBS control implantation from surrounding tissue.H&E sections of 4% silk fibroin hydrogels formed by passive gelation(4P), 4% silk fibroin hydrogels formed by ethanol-enhanced gelation (4E)and 6% silk fibroin hydrogels formed by ethanol-enhanced gelation (6E)all appeared similar, with mild inflammation in all cases characterizedby lymphocytes, macrophages, some neutrophils and fibroblasts (FIG. 10).Cellular infiltration was observed in all sample types with completepenetration in 4P and peripheral ingrowth to a depth of about 100 μm inboth EEG gels with no evidence of cyst formation observed. In all gels,early bioresorption was indicated by implant edge erosion with residualimplant material remaining localized into large lakes. Host integrationof implanted gel had progressed in Day 28 samples of 4E and 6E evidencedby greater cellular ingrowth into the material with complete implantpenetration in 4E samples and robust peripheral ingrowth in 6E samples.The cellular response at this time point was characterized byfibroblasts, lymphocytes and macrophages with the addition of a fewmulti-nucleated giant cells.

TABLE 2 Silk Hydrogel Formulations Group Silk Saline Name ConcentrationEnhancer Component 4E10 4% 90% Ethanol 10% 4R10 90% Ethanol, 1:1 23RGD4RH10 90% Ethanol, 3:1 23RGD 4E25 90% Ethanol 25% 4R25 90% Ethanol, 1:123RGD 4RH25 90% Ethanol, 3:1 23RGD 6E10 6% 90% Ethanol 10% 6R10 90%Ethanol, 1:1 23RGD 6E25 90% Ethanol 25% 6R25 90% Ethanol, 1:1 23RGD6RH25 90% Ethanol, 3:1 23RGD

Day 57 samples of 4E and 6E showed continued host bioresorption of thegel material as there was little residual 4E and while 6E remainedvisible in large, intact lakes, the gel had been completely penetratedwith host tissue. The host response to 4E had dramatically decreased incellularity between Day 28 and Day 57 with very little evidence ofhypercellularity at Day 57 with some scattered macrophages andfibroblasts around the implant site. The pathology of the host responseof 6E was similar to the Day 28 response to 4E, with fibroblasts as thepredominant cell type and scattered lymphocytes, macrophages andmulti-nucleated giant cells. This was viewed as a low-grade, persistent,fibrotic-type inflammatory response to the material.

Samples of 23RGD-enhanced gel exhibited a less robust inflammatoryresponse at the 14 day time point in comparison to non-23RGD-enhancedgel (FIG. 11). This is observed through an appreciable decrease inhyper-cellularity proximal to the gel implant and an accompanyingdecrease in the fragmentation of the implant material. It is possiblethat this improvement in implant integrity is due to a less robustforeign body response by the host animal and it may also be evidencethat there is less mechanical contraction of the implant site, acommonly observed phenomenon with biomaterials including the “RGD”motif. These effects indicate that 23RGD-enhancement of silk gels leadsto a more biocompatible material with better implant outcomes.

In a second trial, intraderm ally-injected samples of silk hydrogel,both ethanol enhanced and 23RGD-ethanol enhanced and relevant controlmaterials were investigated using male Hartley guinea pigs. This wasdone according to protocol#29-05 on file with New England MedicalCenter's Department of Laboratory Animal Medicine (DLAM) and approved bythe Institutional Animal Care and Use Committee (IACUC). Briefly, maleHartley guinea pigs weighing 300-350 g were anesthetized via aketamine/xylazine cocktail injected intramuscularly into the animals'hind legs. The dorsal skin of the guinea pigs was then shaved closelyand swabbed with alcohol, allowed to dry, swabbed with BETADINE®microbicide or Chloraprep (Enturia, Inc., Leawood, Kans.), then drapedwith sterile towels. A 50 μL volume of the desired material was injectedthrough a 26 g needle at six different sites along the left side of theanimal's back. Further injections of an appropriate silk gel controlwere made at the six contralateral sites. Explanation of the silk gelswas performed at 28 days after implantation. Gross observations werecollected semi-weekly regarding implant site appearance. After sampleharvest, gross observations of the implants were conducted and sampleswere processed for histological evaluation. Analysis of histology slideswas provided by a trained veterinary pathologist. Scoring andstatistical analysis was performed as described above.

Table 3 lists the formulations of silk gel, both 23RGD-ethanol-enhancedand ethanol-enhanced developed and assessed intradermally in a guineapig model in a twenty-eight day screen. Although no statisticallysignificant differences were identified, the data for both grossobservations and histology (Tables 4 and 5) indicate a general trendsupporting the previous data that 23RGD-enhancement of gel improvesmaterial biocompatibility. Among sites implanted with silk gel, grossoutcomes varied. Ulceration and hair loss rates were lower in groupswith 25% PBS compared to 10% saline, 6% silk compared to 4% silk andRGDEEG casting as compared to just EEG casting (Table 4). Site rednessrates followed a similar pattern with the exception that RGDEEG samplesinduced more site redness than EEG samples. All silk gels showedevidence of epidermal cyst formation, fibrosis, epidermal hyperplasiaand pronounced inflammation with traces of follicular atrophy in all EEGsamples. Sebaceous cell hyperplasia was present to a limited extent inall formulations with the exception of 6% silk, 10% saline, 1:1 23RGD(Table 5). This is particularly evident in the case of silk gels of 4%silk with 25% saline added and either enhanced with an ethanol-basedenhancer or an 23RGD-ethanol-based enhancer, and more specifically, inthe case of site ulcerations (Table 5). This material indicated strongimprovements with increasing 23RGD concentration in the number of sitesulcerating throughout the course of the trial. These results areindicative that use of 23RGD in conjunction with an ethanol enhancerprovides an improved outcome when compared to an ethanol enhancer alone.

TABLE 3 Silk Hydrogel Formulations Group Silk Saline Name ConcentrationEnhancer Component 4E10 4% 90% Ethanol 10% 4R10 90% Ethanol, 1:1 23RGD4E25 90% Ethanol 25% 4R25 90% Ethanol, 1:1 23RGD 4RH25 90% Ethanol, 3:123RGD 6E10 6% 90% Ethanol 10% 6R10 90% Ethanol, 1:1 23RGD 6E25 90%Ethanol 25% 6R25 90% Ethanol, 1:1 23RGD

TABLE 4 Gross Evaluation of Guinea Pigs Group Site Hair Name RednessLoss Palpability Ulceration 4E10 38% 58% 65% 33% 4R10 57% 49% 67% 33%4E25 28% 34% 49% 28% 4R25 44% 34% 64% 17% 4RH25 50% 23% 66% 6% 6E10 63%52% 68% 33% 6R10 78% 51% 68% 22% 6E25 33% 31% 69% 11% 6R25 56% 30% 68%13% HYLAFORM ™  6% 12% 63% 0% ZYPLAST ™ 17% 10% 52% 0%

TABLE 5 Histological Evaluation of Guinea Pigs Epidermal Cyst EpidermalFollicular Sebaceous Group Name Formation Fibrosis InflammationHyperplasia Atrophy Hyperplasia 4E10 22% 100% 70% 59% 11% 22% 4R10 74%100% 62% 67%  0% 14% 4E25 50% 100% 69% 67% 13% 13% 4R25 29% 100% 39% 62% 0% 14% 4RH25 14% 100% 64% 50%  0% 43% 6E10 44% 100% 70% 56% 11% 33%6R10 25% 100% 63% 38%  0%  0% 6E25 30% 100% 60% 40% 10% 20% 6R25 29%100% 64% 33%  0% 14% HYLAFORM ™  0%  0%  3%  6%  0%  0% ZYPLAST ™  0% 25% 28% 31%  0%  0%

A third trial also used male Hartley guinea pigs to investigateintradermally injected samples of silk hydrogel as described above,comparing samples of 4% and 6% silk, 25% saline 3:1 23RGD-ethanolenhanced silk gels with a collagen-based control material, ZYPLAST™(Allergan Inc., Irvine Calif.) and HYLAFORM™ (Allergan Inc., IrvineCalif.). Explanation of the silk gels was performed at 92 days afterimplantation. Gross observations were collected semi-weekly regardingimplant site appearance. After sample harvest, gross observations of theimplants were conducted and samples were processed for histologicalevaluation. During the course of the 92 day trial, none of the 24implant sites, either 23RGD-ethanol-enhanced hydrogel or ZYPLAST™,ulcerated. Histology revealed that 75% of all ZYPLAST™ sites hadresidual material as did 75% of all 23RGD-ethanol-enhanced silk gelsites (both 4% and 6%). Both materials exhibited very similar chronicphase cellular responses, as the sites were characterized by a mildfibrotic reaction with abundant deposition of collagen in and around theimplant site (FIG. 12). The collagen appears less ordered than does thatin the surrounding dermal reticulum based upon the color density whenviewed with Trichrome staining and also when viewed under polarizedlight. Silk gel sites had similar palpability scores to both controlmaterials but exhibited higher rates of site redness, hair loss andulceration than did ZYPLAST™ and HYLAFORM™. These results not onlyreinforce that 23RGD-ethanol-enhanced silk gel is biocompatible, butalso indicate that it is comparable to collagen biomaterials in terms ofits persistence and long-term behavior in vivo.

ZYPLAST™ exhibited no epidermal cysts, follicular atrophy, or sebaceouscell hyperplasia, though it did show small levels of fibrosis,inflammation and epidermal hyperplasia. Examination of histologicalsections showed residual silk gel material which stained in a mildlyeosinophilic fashion and appeared as large lakes of material at acentral location with smaller masses of material distributed more widelythroughout the reticular dermis (FIG. 13). These smaller masses weretypically surrounded by fibroblasts and macrophages with occasionalmulti-nucleated giant cells present. Eosinophils were located proximalto these smaller masses of implant as well. In general, host response tothe silk fibroin gels was characterized as mildly fibrotic and includedpopulations of fibroblasts, lymphocytes, macrophages, multi-nucleatedgiant cells and eosinophils. Little difference was evident between silkgel types except in terms of the extent of eosinophilia. Largereosinophil populations were observed for 6% as compared to 4% silk gelsand were also observed to increase with RGD concentration in the silkgel samples in both 4% and 6% groups. ZYPLAST™ exhibited strongeosinophilic staining and was distributed as large lakes in thereticular dermis with smaller masses throughout the area.Hypercellularity near the injection site was lessened in ZYPLAST™samples when compared to silk gel. Fibroblasts, lymphocytes,macrophages, multi-nucleated giant cells and eosinophils were presentwith less tendency to localize at the implant periphery. HYLAFORM™samples examined showed many very small masses of material throughoutthe reticular dermis. HYLAFORM™ exhibited no epidermal cysts, fibrosis,follicular atrophy, or sebaceous cell hyperplasia with extremely limitedinstances of inflammation and epidermal hyperplasia. There was noobservable hypercellularity near the implanted material or otherevidence of inflammation at the implant sites.

At day 92, histological evaluation of 4% silk fibroin hydrogel, 3:123RGD, 25% saline (4RH25) samples and ZYPLAST™ samples showed similarmaterial persistence and host response (FIG. 14). Very little implantmaterial remained visible in the dermis of the animals with nohypercellularity present at this time point, evidence of hyperplasia orcellular inflammation. The eosinophils found at day 28 in the ZYPLAST™and silk gel samples were not observed at day 92. Of particularinterest, 4RH25 also exhibited residual disruption to the reticulardermis in the form of an irregular collagen pattern near the implantmaterial. The disorganization of the collagen was seen as a region ofstained collagen seen to be devoid of the typical cross-hatch pattern ofnormal reticular dermis (FIG. 14C). This disorganization was confirmedwhen viewing the histological sections under polarized light with thedisorganized collagen appearing as an interruption in the birefringenceassociated with the surrounding reticular dermis (FIG. 14D).

Example 11 Enhanced Injectable Gel Formulation

Silk hydrogels were prepared as described above in Examples 1-4. Onceprocessed, the gels were sized into coarse or fine particles using asieving step (Table 6). Gel materials were pressed through a 316SSstainless steel wire cloth sieve with a stainless steel spatula and intoclean polystyrene Petri dishes. Sieves with gap sizes of 711 μm×711 μm,295 μm×295 μm, 104 μm×104 μm and 74 μm×74 μm were used. After passingthrough the 74 μm×74 μm gap sieve, the material was considered processedto a “coarse” state. Samples to be processed to a “fine” state werefurther forced through a 43 μm×43 μm sieve in the same fashion. Thissieving was conducted four separate times for each sample type, eachsieving using an approximate quantity of 0.5 mL of gel material.

TABLE 6 Particle sizing Nominal Silk 23RGD Molar Ratio Group MassPercentage with Silk Fineness Name 2% 1:1 Fine 2RF 4% 0 4F 1:1 Coarse4RC Fine 4RF 3:1 RHRF 10:1  4VHRF 8% 1:1 Coarse 8RC Fine 8RF

Samples of silk gel material (N=4 of each type) were evaluated underlight microscopy. Briefly, a 100 mg portion of silk gel or controldevice was massed using an SI-215 Summit series balance. This materialwas loaded into the open back end of a 3 mL syringe using a stainlesssteel spatula. The plunger was replaced in the syringe, an 18 g needlewas attached to the end of the syringe and approximately 900 μL ofultra-pure water was drawn up. This mixture of water and silk gel wasmixed through gentle shaking. After mixing to suspend evenly, a sampleof approximately 30 μL of dilute silk gel was placed on a 75 mm×25 mmsingle frosted, pre-cleaned micro slide (Fisher Scientific Co., Waltham,Mass.) and covered with a 22 mm×40 mm premium cover glass (Corning Inc.,Corning, N.Y.). This sample slide was then be imaged with a microscope.Sample slides were imaged using a System Microscope Model BX41 (Olympus,Melville, N.Y.) in conjunction with a Microscope PC MACROFIRE™ ModelS99831 Camera (Optronics, Goleta, Calif.) and PICTUREFRAME™ 2.1 software(Optronics, Goleta, Calif.). Briefly, slides were scanned for clearlyseparated gel particles using the 4× objective lens and locationsdetermined for a series of 3 representative images of the sample slide.Each of these locations was imaged after first switching the microscopeobjective lens to 10×. Micrograph image files were subjected to analysiswith IMAGE-PRO® Plus 5.1 software (Media Cybernetics, Inc., SilverSpring, Md.). Image files were checked for particle size distribution,average particle size, average aspect ratio, maximum particle size,minimum particle size and standard particle size deviation. Acompilation of the data is presented in Table 7.

TABLE 7 Particle Comminution Data Group Min to Max Object Mean ObjectName Area (μm²) Area (μm²) 2RF 5.33 to 1.32 × 10⁴ 52.43 ± 261.82 4F 5.33to 8.07 × 10³ 27.82 ± 129.34 4RC 5.33 to 8.52 × 10³ 38.41 ± 196.67 4RF5.33 to 5.29 × 10³ 34.12 ± 135.31 4HRF 5.33 to 7.51 × 10³ 40.62 ± 166.614VHRF 5.33 to 3.14 × 10³  35.4 ± 105.43 8RC 5.33 to 8.04 × 10³ 46.57 ±225.43 8RF 5.33 to 2.85 × 10³ 35.26 ± 129.63 ZYPLAST ™ 5.33 to 1.95 ×10³ 22.08 ± 41.71 

Examination of the particles under light microscopy revealed someclumped gel particles which were removed from particle sizing datamanually. Particle sizes ranged from 5.3 to 1.3×10⁴ μm², comparable inrange to commercially available ZYPLAST™ which ranged from 5.3 to1.95×10³ μm². The data also revealed mean particle sizes ranging from27.8 μm² to 52.4 μm², again, comparable to ZYPLAST™ with a mean particlesize of 22.1 μm². These data illustrate that silk gel may besuccessfully comminuted to small and functionally useful particle sizesin a fashion similar to presently utilized injectable gel materials. Thebasic forced-sieving method could easily be replaced with moresophisticated, reproducible methods for purposes of scale-up.

After comminution and blending, samples of silk gel emulsions weresubjected to extrusion force testing. Gel materials prepared asdescribed in Examples 1-4 were blended with appropriate ratios of salinein order to evaluate injection (extrusion) force profiles relative to acontrol material, ZYPLAST™ (Table 8). This was accomplished by massing 5g of gel material in a large weighing boat using an SI-215 balance(Denver Instrument, Denver, Colo.). An appropriate quantity of salinewill be added to constitute the correct volume percentage making theassumption that both the gel material and saline have a density of 1g/mL. This material was then blended to an even consistency using astainless steel spatula and loaded into the back end of a 10 mL syringewith an 18 g needle attached for subsequent use.

TABLE 8 Silk Gel Injection Force Profile Generation Nominal Silk 23RGDMolar Ratio Saline Group Mass with Silk Fineness Content Name 2% 1:1Fine 25% 2RF25 4% 0 25% 4F25 1:1 Coarse 25% 4RC25 Fine  0% 4RFO 25%4RF25 50% 4RF50 3:1 25% 4HRF25 10:1  25% 4VHRF25 8% Fine 25% 6RF25

These samples were tested using an Instron 8511 (Instron Corp., Canton,Mass.) in conjunction with Series IX software and a custom-designedaluminum frame attached to a 100 N load cell (FIG. 15). For the materialtesting, 1 mL of the sample material of interest was loaded into a 1mLgas-tight glass syringe. The sample syringe was mounted in thecustom-designed aluminum frame mounted on the Instron unit and thematerial extruded. The sample was then checked for the force required toextrude the gel at each of 3 strain rates, 10 mm/minute, 50 mm/minute,and 200 mm/minute with total actuator displacement set at 7 mm. A seriesof four tests were run on each material type at each piston displacementrate. Load-displacement data was collected at a frequency of 100 Hz andare presented as the mean ± the standard deviation of the 4 averageextrusion forces experienced of each gel type at each strain rate. Theaverage extrusion force was defined as the average load measured in theplateau region of the load-displacement curve resultant from eachextrusion test. The data were reported as the average amount of forcerequired for extrusion of the sample material and are compiled in Table9 and FIG. 16.

TABLE 9 Average Force (N) to Extrude Silk Gel from 30 g Needle PlungerDisplacement Rate 10 mm/min 50 mm/min 200 mm/min Group Name Ave StdevAve Stdev Ave Stdev 2RF25 0.6 0.0 2.9 0.6 7.3 0.7 4RF25 3.7 2.0 4.5 1.322.4 6.7 4RD25 7.1 3.7 6.7 0.5 25.1 3.9 4RF0 9.5 1.0 28.5 3.1 66.2 10.04RF26 3.2 0.9 7.4 0.6 30.4 5.0 4RF50 1.2 0.2 2.7 0.1 10.1 0.3 4HRF25 2.20.4 8.9 1.0 22.0 0.6 4VHRF25 2.8 1.6 5.2 1.4 14.6 2.1 8RF25 3.6 0.7 10.11.3 29.2 2.4 ZYPLAST ™ 1.6 0.5 18.7 0.7 29.1 1.4

A comparison between milling techniques revealed that there were nosignificant differences between 4RC25 and 4RF25, having averageextrusion forces of 7.1±3.7N and 3.2±0.9N at 10 mm/min, 6.7±0.5N and7.4±0.6N at 50 mm/min, and 25.1±3.9N and 30.4±5.0N at 200 mm/minrespectively (Table 6, FIG. 16A). Both of these formulations differedsignificantly (p≦0.05) from ZYPLAST™ at strain rates of 10 and 50mm/min, which had extrusion forces of 1.6±0.5 N, 18.7±0.7 N, and29.1±1.4 N at 10, 50, and 200 mm/min strain rates.

Data regarding the extrudability of silk gel formulations clearlyillustrated that the addition of saline as a carrier fluid to thecomminuted silk particles offers an improved degree of extrudability,substantially reducing the force necessary to extrude silk gel at allstrain rates. Adding increasing concentrations of saline to thecomminuted silk gels significantly decreased the extrusion forcerequired for silk gels at each strain rate, with gels again exhibitingshear-thickening behavior (Table 9, FIG. 16B). At all strain rates, 4RF0required significantly more force to extrude than 4RF25, which in turnrequired significantly more than 4RF50. At a strain rate of 10 mm/min,4R0, 4R10, and 4R25 showed a significant decrease (p≦0.05) in extrusionforce with increasing PBS concentration, having average forces of9.5±3.1 N, 6.1±0.5 N, and 4.7±0.7 N respectively (Table 9). At 50mm/min,these relationships were more pronounced with average extrusion forcesof 14.0±0.9 N, 5.4±0.7 N, and 3.9±0.2 N respectively and all differedsignificantly (Table 6, FIG. 16). At 200 mm/min, the trend remained asaverage extrusion forces were 26.4±4.5 N, 10.6±1.6 N, and 6.4±0.5 Nrespectively with 0% PBS differing significantly from the other twogroups. Samples of 6R25 had an average extrusion force of 29.3±4.8 N at10 mm/min, significantly higher than 4R25 (Table 9). At 50 mm/min and200 mm/min, the force to extrude the 6R25 was greater than 80 N, causingthe test to abort in order prevent damage to the load cell.

The data also illustrate that use of very low concentrations of silk mayimprove the extrudability of gel relative to higher concentrations as inthe case of 2RF25 as compared to 4RF25 and 8RF25. Increasing theconcentration of silk in the comminuted silk gels increased theextrusion force required for silk gels at each strain rate, withsignificant increases between 2RF25 and both 4RF25 and 8RF25 at 10mm/min and 200 mm/min (Table 9, FIG. 16C). All groups differedsignificantly at the 50 mm/min strain rate and gels continued to exhibitshear-thickening behavior, seen in the increased extrusion forcesassociated with increased strain rates. At 10 mm/min 2RF25 and 8RF25required 0.6±0.0 N and 3.6±0.7 N respectively, at 50 mm/min theyrequired 2.9±0.6 N and 10.1±1.3 N, and at 200 mm/min 7.3±0.6 N and29.2±2.4 N.

The data further indicated that use of 23RGD to enhance the silk gelmaterial did not appreciably impact the force necessary to extrude silkgel formulations. Adding increasing concentrations of RGD did not have aconsistent effect upon the extrusion force necessary for the gelmaterials (Table 9, FIG. 16D). At a 10 mm/min strain rate there were nosignificant differences between 4F25 at 3.7±2.0 N, 4R25, 4HR25 at2.2±0.4 N, and 4VHR25 at 2.8±1.6 N. At a 50 mm/min strain rate 4HR25 wassignificantly higher than all other extrusion forces at 8.9±1.0 N ascompared to 4F25 at 4.5±1.3 N, 4R25, and 4VHR25 at 5.2±1.4N. At a 200mm/min strain rate 4HR25 at 22.0±0.6 N was significantly higher thanonly 4VHR25 at 14.6±2.1 N as compared to 4F25 at 22.4±6.7 N and 4R25.

Lastly, the data showed that silk gels blended with saline had verysimilar extrudability to ZYPLAST™, a material already proven to bereadily handled as an injectable material. Based upon this data it isbelieved that through careful manipulation of the carrier speciesassociated with the silk gel, modulation of silk concentration, andcontrol of particle size, silk gel materials may be made to behave as areadily injectable material.

These results indicate that silk gels may be comminuted to a particlerange of about 25-50 μm² in cross-sectional area. Silk gels may becomminuted to a size similar to ZYPLAST™. Silk gel particle size can bedecreased by increasing silk concentration or by changing the method ofcomminution. Increasing concentrations of RGD did not develop a cleartrend in silk particle size. Silk gels may be extruded at a relevantstrain rate of 50 mm/min at a force comparable to or less than ZYPLAST™.Silk gel extrusion force may be decreased by adding increased quantitiesof saline carrier or decreased concentrations of silk in the originalgel. Changes of comminution method attempted in this study did notsubstantially affect the amount of force necessary for silk extrusion.Increasing concentrations of RGD did not develop a clear trend in silkgel extrusion force.

Example 12 Silk Gel Precipitates

The silk gel precipitate materials outlined in Table 10 were generatedfor analysis. Silk solution of the specified concentration was generatedusing the stock solution of 8% (w/v) aqueous silk and diluting withpurified water (Milli-Q purified). 23RGD/ethanol accelerant was preparedby generating a solution of ethanol and purified water, then dissolvingthe specified 23RGD quantity by vortexing. Silk precipitates weregenerated by directly adding the specified volume of accelerant solutionto that of silk solution in 50 mL centrifuge tubes, shaking once to mixand allowing the mixture to stand for 5 additional seconds before addingabout 45 mL purified water to halt the gelation process. This materialstood for 24 hours under ambient conditions and was then strainedthrough stainless steel cloth with 150 μm×150 μm pores to recoverprecipitates. These precipitates were rinsed twice for 24 hours in 50 mLof purified (Milli-Q) water at room conditions, strained a final timeand used for evaluation.

TABLE 10 Silk Gel Precipitate Types Generated Initial Silk Solution23RGD/ethanol Accelerant Silk Accelerant Final Precipitate Silk SolutionEthanol 23RGD Solution Final Silk RGD:Silk Group Concentration VolumeConcentration Concentration Volume Concentration Molar Name (mg/mL) (mL)(%) (mg/mL) (mL) (mg/mL) Ratio BASE 80 1 90 2.45 1 40 5.0 RHI 80 1 904.90 1 40 10.0 RVLO 80 1 90 0.49 1 40 1.0 RLO 80 1 90 1.47 1 40 3.0 SCLO80 1 90 2.45 1 30 6.7 SCVLO 80 1 90 2.45 1 20 10.0 ECLO 80 1 80 2.45 140 5.0 ECVLO 80 1 70 2.45 1 40 5.0 AVHI 80 0.67 90 2.45 1.33 27 10.0AVLO 80 1.33 90 2.45 0.67 53 2.5

Samples of gel were examined under low-vacuum conditions (˜1 Torr) on aQuanta 200 (FEI Co., Hillsboro, Oreg.) environmental scanning electronmicroscope with images collected at magnifications of 200×.Representative images were taken to illustrate surface topographycharacteristics of silk precipitate samples (FIG. 17). All silkprecipitate types appeared similar under ESEM analysis. Each sampleexhibited a mixture of both granular and filamentous regions withoccasional appearance of large, contiguous masses of smooth material.

Example 13 Silk Gel Precipitate Massing

Silk precipitate samples, as described in Example 12, were isolatedafter rinsing by straining through stainless steel wire cloth with apore size of 104 μm×104 μm and gently blotted with a clean, lint-freewipe. Samples were massed to the nearest 0.01 mg using an S-215 balance(Denver Instrument, Denver, Colo.). These samples were frozen to −80° C.for 24 hours and placed into a Labconco lyophilizer unit (LabconcoCorp., Kansas City, Mo.) for 96 hours to remove all water content. Theprecipitate residual solids were massed again and the dry mass fractionin the samples determined. One-Way analysis of variance (ANOVA) was usedto test for significant differences caused by changing silkconcentration, 23RGD concentration and accelerant volume. A Student'st-test was used to test the significance of differences resulting fromaltered ethanol concentrations.

Increasing silk fibroin concentration increased precipitate dry masswith Increasing the percentage of ethanol in the accelerant solutionalso increased dry mass of the precipitates with ECVLO produced onlytrace quantities of precipitate (visible, but not recoverable inmeasurable quantities).

Increasing accelerant volume significantly increased precipitate drymass as AVHI was significantly greater than both AVLO and BASE (p≦0.05,FIG. 18A). For example, AVHI (18.02±3.9 mg) was significantly greaterthan both AVLO (7.37±1.33 mg) and BASE (11.07±2.86 mg). Increasingconcentrations of 23RGD in the accelerant also increased the dry mass ofprecipitate with BASE and RHI both significantly higher than RVLO at(FIG. 18B). Fore example, BASE at 11.07±2.86 mg, RHI at 15.61±3.62 mg,and RMED at 10.2±1.42 mg were all significantly higher than RLO at1.9±0.6 mg. Increasing silk fibroin concentration increased precipitatedry mass with BASE being greater than SCLO and significantly greaterthan SCVLO (FIG. 18C). For example, BASE was greater than SOLO at7.84±1.49 mg and significantly greater than SCVLO at 4.15±1.0 mg.Increasing the percentage of ethanol in the accelerant solution alsoincreased dry mass of the precipitates with BASE producing significantlymore than ECLO (FIG. 18D). For example, BASE produced significantly morethan ECLO at 2.8±0.91 mg. ECVLO produced only trace quantities ofprecipitate (visible, but not recoverable in measurable quantities).These results indicate that greater concentrations of reactants (i.e.,accelerant solution, RGD, silk and ethanol) all increased the quantityof precipitant resultant.

The percent water in silk precipitates was determined as the percentageof mass lost after silk precipitates of each formulation types weresubjected to a lyophilization step. Increasing the volumetric fractionof accelerant added to make silk precipitates did not significantly(p≦0.05) affect the dry mass fraction of the resultant precipitates(FIG. 19A). For example, AVLO at (85.57±2.32%, BASE at 88.99±0.8%, andAVHI was 86.83±1.95%. Increasing concentrations of 23RGD in theaccelerant showed a significant increase in dry mass percentage withRVLO significantly less than RLO, RHI, and BASE (FIG. 19B). For example,RLO at 95.01±1.76% retained significantly more water than RMED at86.52±2.67%, RHI at 88.39±0.98%, and BASE. Increasing concentrations ofsilk fibroin did not result in a clear trend although SCLO wassignificantly greater than both SCVLO and BASE (FIG. 19C). For example,SCLO at 80.77±1.97% was significantly less than both SCVLO at86.94±1.98% and BASE. Increasing the percentage of ethanol in theaccelerant solution significantly decreased the dry mass percentage withECLO compared to BASE (FIG. 19D). For example, ECLO at 86.97±1.16%compared to BASE. In summary, greater concentrations of reactants (i.e.,accelerant solution, 23RGD, silk and ethanol) increased the quantity ofresultant precipitate. It is also of interest that there weresignificant differences between the dry mass fractions of BASE and bothRVLO and ECLO, possibly indicating different protein structures. Thesediffering protein structures might be more hydrophobic than BASE in thecase of ECLO and more hydrophilic in the case of RVLO. These propertiesmight used to affect the stability of the gels in an in vivo environmentwith more hydrophilic materials being more readily bioresorbed by thehost while more hydrophobic materials prove more resistant.

In examining the percent of water in the precipitates it is ofparticular interest that there were significant differences between BASEand both RLO and ECLO. This may result from structural motifs differentthan other precipitate types generated by RLO and ECLO. With respect toECLO, it has a greater proportion of β-sheet structure than BASE andwould be expected to entrain less water. However, the differenceobserved between RLO and base is difficult to explain. RLO has a greaterextent of β-sheet structure with less α-helix and random coil motifsthan BASE, yet it entrains a greater quantity of water. In fact, thissame trend is seen when comparing RLO to RMED, BASE, and RHI. Thesituation is further confounded in examining the relationship betweenthe initial secondary structures of RMED, BASE and RHI, as all initiallyexhibit greater quantities of α-helix and random coil than RLO, yet allentrain significantly less water. SCLO samples also had a significantlyhigher dry mass percentage as compared to BASE and SCVLO sample with noclear trend or reason for this occurrence. These data indicate thatthere may be a structural difference in these precipitates not apparentin the secondary structure of the materials which is affecting themanner in which the precipitates associate with water. It may be thecase that the RGD bound to these precipitates has altered in somefashion the manner in which the silk molecules are presented to water,enhancing their ability to associate with it.

Example 14 Gel Precipitate FTIR Spectrum Capture

Gel precipitates of each type, as described in Example 12, were analyzedby attenuated total reflectance Fourier-transform infrared (ATR-FTIR)spectroscopy using a Bruker Equinox 55 spectrophotometer (Bruker Optics,Inc., Billerica, Mass.) coupled with a Pike MIRACLE™ germanium crystal(PIKE Technologies, Madison, Wis.). Sample ATR signal spectra wereobtained by performing a 128-scan integration. Resolution was set to 4cm⁻¹ with a 1 cm⁻¹ interval from a range of 4000 to 400 cm⁻¹. FTIRspectra of pure water were also collected and subtracted manually fromthe gel spectra to remove confounding water signal at a ratio conduciveto flattening the region between 1800 cm⁻¹ and 1700 cm⁻¹ on thespectrum. After subtraction, the Amide I bands (1700-1600 cm⁻¹) ofrepresentative spectra were evaluated against characteristic peakscommonly accepted to be associated with secondary protein structures.

Examination of the silk precipitates under FTIR revealed that increasingthe volumetric ratio of accelerant added to the silk solution had littleeffect on their protein secondary structure (FIG. 20A). AVLO, BASE, andAVHI all exhibited similar characteristics with characteristic peaksaround 1624 cm⁻¹ and a toe region at 1698 cm⁻¹ indicating a predominanceof β-sheet and β-turn structure respectively. Each sample also exhibitedadditional structures at 1677 cm⁻¹, 1663 cm⁻¹, 1654 cm⁻¹ and 1645 cm⁻¹denoting additional interspersed β-sheet, β-turn, α-helical and randomcoil conformations respectively. Increasing concentrations of 23RGD inthe accelerant decreased β-sheet stability indicated by a peak shiftfrom ˜1621 cm⁻¹ in RVLO to ˜1624 cm⁻¹ in RLO (FIG. 20B). Furtherincreasing the concentration of 23RGD in BASE and RHI caused thisweakened β-sheet again accompanied by an increase in higher signalvalues in the 1654 cm⁻¹ and 1645 cm⁻ranges, indicating increased randomcoil and α-helical constituents. Otherwise, RVLO, RLO, BASE, and RHIrevealed similar structures with dominant peaks in the 1620 cm⁻¹ rangeand a toe region at 1698 cm⁻¹ with additional structures at 1654 cm⁻¹and 1645 cm⁻¹. Increasing concentrations of silk fibroin had littleperceptible effect on protein secondary structure (FIG. 20C). Thespectra for SCVLO, SCLO, and BASE each exhibited similar characteristicpeaks around 1624 cm⁻¹ with toe regions at 1698 cm⁻¹ indicating apredominant β-sheet structure with additional α-helical and random coilconformations interspersed. Increasing the percentage of ethanol in theaccelerant solution resulted in less evidence of α-helical and randomcoil conformations indicated by a decrease in the signal between 1670cm⁻¹ and 1630 cm⁻¹ in both ECLO and BASE samples relative to ECVLO (FIG.20D). This decrease in α-helical and random coil is accompanied by anincrease in β-sheet structure.

Substantial similarity existed between all groups except for RVLO andECVLO, which each differ from BASE formulation. Each of these materialtypes exhibited a different secondary structure from both each other andfrom BASE, reinforcing the trend observed previously in the percent drymass of the precipitates. Higher concentrations of 23RGD yielded lessorganized β-sheet structures and lower concentrations of ethanol yieldedgreater quantities of α-helix and random coil motifs. It is possiblethat used in conjunction with one another, these two phenomena could beadjusted to develop silk structures resulting from silk solutions in anyof a variety of different protein conformations. These conformationscould, in turn, be tailored based upon the desired ultimate bulkproperties of the silk material.

It is expected that higher β-sheet components might provide the gel withgreater resistance to bioresorption and compressive loading, while atthe same time, making the material more rigid.

Example 15 Congo Red Staining of Gel Precipitates

Silk precipitate samples were stained with 100 μM Congo red in purifiedwater. Silk precipitate samples weighing 5-10 mg were vortexed with 500μL of this solution for 15 seconds, allowed to stand at room temperature(˜20-24° C.) for 10 minutes, then centrifuged at 16,000 g (RCF) for 10minutes. The supernatant was discarded and the pellet re-suspended byvortexing for 30 seconds in 1 mL of purified water. The process ofsoaking, centrifugation, aspirating and rinsing was repeated 3 times.The final pellet was removed, smeared on a glass microscope slide, andimaged under white and polarized light using a Microscope PC MACROFIRE™Model S99831 Camera (Optronics, Goleta, Calif.) and PICTUREFRAME™ 2.1software (Optronics, Goleta, Calif.) and a System Microscope Model BX41(Olympus, Melville, N.Y.).

None of the silk precipitate types exhibited the emerald luminescencetypically associated with amyloid fibrillar structures (FIG. 21). Allprecipitate types did exhibit bright white luminescence, indicative of arobust crystalline structure. The extent of this brightness does notappear to vary substantially by formulation, but only by sample quantityon the slide. Based on these results, it is unlikely that any of theseprecipitate types is amyloid in nature, a positive sign, as amyloidfibrils are associated with a number of negative pathologies in humans.

Example 16 23RGD Quantification in Gel Precipitates by HPLC

The amount of 23RGD bound to silk precipitates was quantified byanalyzing lyophilized samples. The 23RGD was removed by incubating thesamples for 4 hours in a dissolving buffer, then centrifuging on anEppendorf 5415C (Eppendorf North America Inc., Westbury, N.Y.) at 16,000g (RCF) for 30 minutes and the supernatant collected. This supernatantwas then centrifuged in identical fashion and the final supernatantcollected for HPLC analysis using a PerkinElmer Series 200 (PerkinElmer,Waltham, Mass.). The 23RGD peak areas from each curve were comparedagainst a standard curve. 1-Way ANOVA was used to test for significantdifferences caused by changing silk concentration, 23RGD concentration,and accelerant volume. A Student's t-test was used to test thesignificance of differences resulting from altered ethanolconcentrations.

Increasing the quantity of 23RGD/ethanol accelerant added resulted in asignificant increase (p≦0.05) in 23RGD:silk ratio for both BASE and AVHIas compared to AVLO (FIG. 22A). For example, BASE at 8.7±0.6 and AVHI at10.5±1.2 were significantly increased as compared to AVLO at 5.2±1.8.Increasing the quantity of 23RGD in the accelerant solution resulted insignificant increases in 23RGD:silk ratio for each of RVLO, RLO, BASE,and RHI relative to each other (FIG. 22B). For example, RLO at 1.1±0.2,RMED at 6.95±0.49, BASE and RHI at 10.7±0.8 relative to each other.Changing the starting concentration of silk in solution prior toprecipitation did not affect 23RGD:silk ratio as those in SCVLO, SCLO,and BASE did not differ significantly (FIG. 22C). For example, SCVLO at11.0±0.4, SCLO at 9.9±1.8, and BASE did not differ significantly.Decreasing the ethanol content in the accelerant did not produce asignificant effect as observed by comparing ECLO and BASE (FIG. 22D).

Reviewing this data in light of the precipitate dry massing data, noneof the conditions explored resulted in isolation of silk (˜10-35%precipitated) nor 23RGD (˜5-30% precipitated) as limiting reagents inthe reaction. Precipitate samples generated at a calculated 10:123RGD:silk ratio consistently generated a “correct” molecular bindingratio. In the case of AVHI, this runs contrary to the trend of bound23RGD concentrations being approximately double the projected values asindicated by AVLO and BASE (about 5:1 and about 9:1, respectively). Thismight be explained by saturation of the silk with 23RGD in the case of10:1 23RGD precipitates. This is further reinforced by the behavior ofSCVLO and 0.6S 3R 10:1, both of which were produced using 2.45 mg/mL23RGD in 90% ethanol as the AVHI was. Both materials projected to havegreater than 10:1 ratios of bound 23RGD (20:1 and 13.4:1, respectively)based on the behavior of AVLO and BASE, but which both reached onlyabout 10:1 ratios. RHI, generated using a 4.5 mg/mL 23RGD concentrationin the accelerant which conceivably should have been high enough toinduce the postulated dimeric 23RGD reached only the expected 23RGDratio of about 10:1 not the postulated 20:1.

Few of the silk precipitates entrained a molar ratio similar to what wasinitially calculated (FIG. 23). Four groups, SCVLO, AVHI, RHI, and RLOcontained ratios similar to their calculated values of RGD per mole ofsilk. The six remaining groups contained ratios substantially greaterthan their calculated values. In the cases of AVLO, BASE, RMED, andSCLO, the RGD quantities were about 2-fold greater than expected.Although not wishing to be limited by theory, this greater observedmolar ratio may be indicative of the formation of a RGD bi-layer. It maybe the case that either micelles or lamellar structures of RGD existedin the 90% ethanol solution prior to addition to the silk, uponcontacting the aqueous phase, micellar stability was disrupted. As aresult, a bi-layer of RGD was formed at the solution interface, wherethese molecules began to interact with the silk molecules. The RLOsamples were made with a RGD concentration of 0.49 mg/mL in theaccelerant, the lowest used in this study and potentially within thesolubility range of RGD in 90% ethanol. RMED samples used 1.47 mg/mL andmost other formulations were made with a RGD accelerant concentration of2.45 mg/mL, above the RGD concentration at which dimerization becamefavorable in the solution. Further highlighting the possibility of RGDdimerizing in the ethanol solution is the behavior of ECLOprecipitation. The RGD concentration remains 2.45 mg/mL as with BASE andAVLO but the water concentration in the accelerant is increased to 20%and results in a binding of about 1.5-fold the expected total of RGD.This may be due to a decreased driving force for RGD bi-layer formationat the solution interface caused by the lower ethanol content. Thismight in turn cause disruption to fewer micellar structures in theinitial accelerant solution. It could also be explained by alteredmicellar structure, varying between a single peptide layer and amulti-lamellar structure depending upon the concentrations of water andethanol in the accelerant phase.

Precipitate samples generated at a calculated 10:1 RGD:silk ratioconsistently generated a “correct” molecular binding ratio. In the caseof AVHI, this runs contrary to the trend of bound RGD concentrationsbeing approximately double the projected values as indicated by AVLO andBASE (about 5:1 and about 9:1 respectively). It is possible that thismight be explained by saturation of the silk with RGD in the case of10:1 RGD precipitates. This is further reinforced by the behavior ofSCVLO and 0.6S 3R 10:1, both of which were produced using 2.45 mg/mL RGDin 90% ethanol as was AVHI. Both materials projected to have greaterthan 10:1 ratios of bound RGD (20:1 and 13.4:1 respectively) based onthe behavior of AVLO and BASE, but which both reached only about 10:1ratios. RHI, generated using a 4.5 mg/mL RGD concentration in theaccelerant which conceivably should have been high enough to induce thepostulated dimeric RGD, reached only the expected RGD ratio of about10:1 not the postulated 20:1. This may be attributed to the mode ofbinding between the silk molecules and the RGD molecules. It is expectedthat RGD will bind through a hydrophobic association mechanism anddespite the largely hydrophobic sequence of silk, it may be possiblethat there are approximately 5 sites which offer preferable RGD bindingstability. This presumption stems from the apparent saturation at 10:1RGD molecules per molecule of silk. Dependent upon the nature of RGDself-association at the solution boundary, it may be a case where singleRGD molecules or RGD dimers bind to these sites.

There are a series of properties further indicating the possibility of aspecific molecular assembly interaction between the silk and 23RGDaccelerant. Conspicuously, that 23RGD does localize to the precipitatesin a greater-than-calculated ratio but that it binds at intuitiveconcentrations which can be related quickly to the initially calculatedmolar ratios. The fact that this occurs without fully depleting eitherthe 23RGD or the silk fibroin molecules is of further interest. The FTIRdata also indicated that use of 0.49 mg/mL 23RGD in RVLO precipitatesinduced formation of distinctly different structures than use of 2.45mg/mL in BASE or 4.9 mg/mL in RHI which appeared similar to each other.RMED precipitates generated with 1.47 mg/mL of 23RGD containedcharacteristics of both RVLO and BASE/RHI material spectra. FTIRindicated a different structure from a 2.45 mg/mL of 23RGD in 70%ethanol accelerant in the case of ECVLO. These outcomes were bothreinforced in examining the percentage of dry mass from the resultantprecipitates (though ECLO is used to illustrate the trend in 23RGDsolubility in ethanol solution instead of ECVLO). Both of these assaysindicate the formation of different precipitate structures based uponthe extent of 23RGD saturation in the ethanol solution, conceivablyresulting from dimeric 23RGD binding or monomeric 23RGD binding.

This phenomenon likely results from the amphiphilic nature of 23RGD andthe varied chemistry of the solution phase between heavily ethanolic andheavily aqueous. It is possible that the hydrophilic ends of two 23RGDmolecules associate in the 90% ethanol solution, exposing the twohydrophobic ends to solution. Addition of this accelerant solution withdimeric 23RGD causes rapid association of the exposed hydrophobic endsof the 23RGD with hydrophobic domains of the silk molecules, rapidlyprecipitating them. This process occurs until the 90% ethanol accelerantsolution is sufficiently diluted with the aqueous silk solution to causethe dimeric assembly of the 23RGD molecules to no longer be favorable,as a result stopping precipitation. Based upon the apparent saturationat about 10 for 23RGD:silk ratio, there may also be a maximum of 5binding sites for the 23RGD dimer per molecule of silk. This knowledgemay be used to bind specific quantities of 23RGD to silk, while at thesame time dictating silk gel structure and resultant behavior.Additionally, this method may also potentially be applied to otheramphiphilic peptides of interest during their integration into a silkgel material.

These results indicate that silk precipitate quantity may be increasedby increasing the quantity of any reactant in the RGDEEG system. Silkprecipitates occurring during RGDEEG gelation are unlikely to beamyloid. Silk precipitate β-sheet structure may be increased by higherconcentrations of ethanol accelerant or lower concentrations of RGD. RGDmolecules may self-associate into micelles, lamellar structures, ordimers when placed into a strongly ethanolic solution, in turn,assembling with silk in a dimeric fashion during RGDEEG gelation. Silkmolecules may become saturated with RGD once they have bound about 10molecules. Silk precipitate structures may be altered by changing RGDconcentrations added, though the extent and nature of these changesremains unclear, as they are not perceptible in material secondarystructure. These altered structures may account for otherwiseunexplained increased appearance of α-helix and random coil motifs athigh RGD concentrations in precipitates. These altered structures mayaccount for otherwise unexplained increased resistance to proteolyticbioresorption of α-helix and random coil motifs at high RGDconcentrations in precipitates

Example 17 Enzymatic Bioresorption of Gel Precipitates

A single sample of precipitate types selected for distinctly differentbehaviors from BASE in the previously listed assays including RVLO, RLO,BASE, RHI, ECLO, 0.65 3R 5:1 weighing approximately 60 mg were massedusing an S-215 balance. These samples were placed in a solution ofProtease Type XIV from Streptomyces griseus (Sigma catalog no. P-5147)in phosphate buffered saline (PBS) was generated at a concentration of0.3 mg/mL (activity was 4.5 U/mg) at a ratio of 1 mL of proteasesolution per 100 mg of silk precipitate. The gel and protease solutionwere incubated for 24 hours at 37° C. with no mechanical mixing. After24 hours, the residual precipitate was isolated by straining throughstainless steel cloth as before and the specimens analyzed by FTIR asdescribed.

Accelerant quantity added did not substantially affect the bioresorptionbehavior of the materials as BASE, AVHI and AVLO all featured decreasedlevels of α-helix and random coil motifs (FIG. 23A). This decrease wasslightly larger in the case of AVLO which also featured a peak shiftfrom 1624 cm⁻¹ to 1622 cm⁻¹, indicating a more stable β-sheet structure.The 23RGD concentration did not appear to affect bioresorption behaviorof the materials either as RVLO, RLO, BASE and RHI all showed decreasedin α-helix and random coil motifs, though a greater portion of α-helixand random coil remained intact in RHI (FIG. 23B). However, a greaterportion of α-helix and random coil remained intact in RHI at Day 2relative to the other samples. Silk concentration did not substantiallyaffect the bioresorption behavior of the materials as BASE and SCLOexhibited decreased levels of α-helix and random coil motifs andfeatured slight peak shifts from 1624 cm⁻¹ to 1623 cm⁻¹ (FIG. 23C).

Despite differences in initial structures, all precipitate typesbioresorbed in a similar fashion with α-helix and random coil motifsdegraded preferentially to β-sheet. Only increasing the concentration of23RGD, as in the case of RHI, appeared to have any appreciable effect onthe final secondary structure of the precipitates. This appears to be acase where there is simply more α-helix and random coil structure uponinitial formation of these materials and they take more time to degradeto a similar extent of β-sheet structure as the other formulations. Useof this knowledge in conjunction with an ability to manipulate thesecondary protein structures of these materials could lead tobiomaterials with very specific lifetimes in vivo.

Example 18 Composition Comprising Silk Fibroin Hydrogel Particles andMatrix Polymer

Silk fibroin hydrogels were cast according to the methods describedabove in Examples 1-4. A silk hydrogel consisting of 6% silk fibroin bydry mass percent with a 23RGD molar ratio of 1:1 with silk molecules wasgenerated for particle comminution. This material was subjected to theforced sieving method described above to generate silk gel particlesranging nominally between 0.1 μm² and 5 μm². These materials wereblended with crosslinked hyaluronan at various volumetric ratios forevaluation as a potential filler material. The blends were made atvolumetric percentages of 5% silk fibroin hydrogel with 95% hyaluronan,25% silk fibroin hydrogel with 75% hyaluronan, 50% silk fibroin hydrogelwith 50% hyaluronan.

Example 19 Composition Comprising Silk Fibroin Hydrogel Particles andMatrix Polymer

The composition described above in Example 18 are modified in terms ofthe 23RGD component (0 to 3:1), silk fibroin concentration (1% to 8%),particle size (0.1 μm² to 500 μm²), and silk format (may be silksolution intermediate mentioned above added directly to hyaluronan).Materials also vary in the percent composition of silk hydrogel inhyaluronan between 0.1% and 99.9% depending upon application and desiredmaterial properties.

Example 20 Extrudability Characteristics of Composition Comprising SilkFibroin Hydrogel Particles and Matrix Polymer

To assess the extrusion force necessary to inject a compositiondisclosed herein through a needle, the dermal fillers comprising silkfibroin hydrogel particles in Table 11 were compared with JUVÉDERM®Ultra Plus (Allergan, Inc., Irvine Calif.), a crosslinked hyaluronandermal filler sample lacking silk fibroin hydrogel particles (6PUR00).The extrusion force test was performed by measuring the force necessaryto extrude a hydrogel through a 27 gauge or 30 gauge needle using a 0.8mL syringe.

To prepare the compositions listed in Table 11, a 6% silk fibroinhydrogel was prepared according to Examples 1-4. Some of it was used ascomponent for the filler formulation, and another batch was mixed with25% (v/v) PBS (saline buffer) according to Example 9, for a final silkfibroin content of around 4.5%. These hydrogels were mixed with acommercial crosslinked hyaluronan in various proportions using amechanic mixer. This hyaluronan hydrogel had the followingcharacteristics: 24 mg/g sodium hyaluronate (with an average molecularweight of 3,000,000 Da before crosslinking), a degree of crosslinking of5%-6% (crosslinker: 1,4-butanediol diglycidyl ether). Table 11 lists theformulations prepared by mixing silk fibroin hydrogels with hyaluronangels in 100 g pots by using several homogenization cycles of 1 minute at3500rpm in the mixer. The pH was adjusted to about 7.0 by addition ofsmall volumes of a diluted sodium hydroxide solution betweenhomogenization cycles.

TABLE 11 Examples of filler formulations containing silk fibroin andcrosslinked hyaluronan Composition Silk Component Final Silk FibroinFinal HA Name Silk Fibroin Hydrogel Weight % Concentration Concentration6PUR00 no silk (blank)  0% 0 mg/g 24.0 mg/g 6PUR05 6% silk fibroin gel 5% 3.0 mg/g 22.8 mg/g 6PUR25 25% 15.0 mg/g 18.0 mg/g 6PUR50 50% 30.0mg/g 12 mg/g 6PBS05 4.5% silk fibroin gel  5% 2.25 mg/g 22.8 mg/g 6PBS25(made from 6% gel + 25% 11.25 mg/g 18.0 mg/g 6PBS50 25% (v/v) salinebuffer) 50% 22.5 mg/g 12 mg/g 6PBS75 75% 33.75 mg/g 6 mg/g

Analysis of these compositions indicate that compositions comprisingabout 5% to about 50% silk fibroin hydrogel particles exhibitedextrudability characteristics similar to a composition comprising 0%silk fibroin hydrogel particles. For example, at a plunger displacementrate of about 13 mm/min, compositions comprising 0% to about 50% silkfibroin hydrogel particles all exhibited an extrusion force of about10N. Similarly, at a plunger displacement rate of about 50 mm/min,compositions comprising 0%, about 5%, and about 50% silk fibroinhydrogel particles all exhibited an extrusion force of about 17N, withcompositions comprising 25% silk fibroin hydrogel particles exhibitingan extrusion force of about 20N.

Example 21 In Vivo Evaluation of Composition Comprising Silk FibroinHydrogel Particles and Matrix Polymer

To examine the in vivo effects of the compositions disclosed herein, thecompositions disclosed in Table 12 were subcutaneously injected intoSprague Dawley rats.

To prepare the compositions listed in Table 12, a 8% silk fibroinhydrogel was prepared according to Examples 1-4. The silk fibroinhydrogel was milled into particles of a mean cross-sectional area ofabout 1 μm and blended with the saline component indicated in Table 12.Saline was added by first mixing by spatula into a bulk of silk fibroinhydrogel, then shearing 60 times through a 1.5 mm orifice. After salineblending, the material was sterilized by gamma irradiation at a dose of25-40 kgy. The sterilized silk fibroin hydrogel particles were blendedwith JUVÉDERM® Ultra Plus (Allergan, Inc., Irvine Calif.), a hyaluronandermal filler, in ratios according to Table 12 immediately prior tosurgery. Blending was conducted by means of shearing back and forthbetween a pair of syringes connected by a stopcock until combined gelappearance was uniform.

TABLE 12 Examples of filler formulations containing silk fibroin andcrosslinked hyaluronan Silk Fibroin Hyaluronan Saline Silk FibroinSST:Silk Group Sample Name Component Component Component Percent SilkRatio 1 JUVÉDERM ® Ultra Plus N/A 100%  N/A N/A N/A 2  5% Base SilkFibroin  3.75% 95% 1.25% 6% 1:1 molar 3 25% Base Silk Fibroin 18.75% 75%6.25% 6% 1:1 molar 4 50% Base Silk Fibroin  37.5% 50% 12.5% 6% 1:1 molar5 75% Base Silk Fibroin 56.25%  5% 18.75%  6% 1:1 molar 6 Base SilkFibroin   75%  0%   25% 6% 1:1 molar

Eight male Sprague Dawley rats weighing 250-275 grams, acclimated forone week at the animal facility prior to surgery, were anesthetized with4% isoflurane and maintained at 1-2% isoflurane on a heated pad. Theback of the animal was shaved and cleaned with alcohol. The animals wereinjected at four different sites on the back with a volume of 50μL/injection. Compositions were distributed across multiple animals in asuccessive and cyclical pattern. Material Group 1 will be injected intoAnimal 1, Site 1; Material Group 2 into Animal 1, Site 2; Material Group3 into Animal 1, Site 3; Material Group 4 into Animal 1, Site 4;Material Group 5 will be injected into Animal 2, Site 1, Material Group6 will be injected into Animal 2, Site 2; Material Group 1 will beinjected into Animal 2, Site 3; Material Group 2 will be injected intoAnimal 2, Site 4, etc. A total of 24 sites will be injected in 6 rats.On Day 42, animals were euthanized via carbon dioxide asphyxiation andall sample sites were identified, harvested, and prepared for subsequentsectioning and staining.

Material cross-sections mounted on slides were stained with H&E andCD-68 according to standard methods (FIG. 24 and FIG. 25). It wasobserved that all sample types infiltrated the animal dermis andappeared as lakes of material. Pure JUVÉDERM® Ultra Plus controlelicited a minimal extent of cellular response, very consistent withambient cellularity in the surrounding tissue (FIG. 24). The extent ofcellular infiltrate and total presence was increased by adding silkfibroin hydrogel particles to the HA dermal filler (FIG. 24). Base silkfibroin hydrogel particle material by comparison, exhibited significantcellular response which tended to occur circumferentially around smalleragglomerated lakes of material (FIG. 24). Staining with CD-68 revealedthe presence of small populations of CD-68+ cells in all silk fibroinhydrogel particle containing samples but in none of the pure JUVÉDERM®Ultra Plus control samples (FIG. 25). This suggests increased macrophageactivity in the silk fibroin hydrogel particle-containing samples ascompared to the pure JUVÉDERM® Ultra Plus control.

The results reveal the potential for increasing the extent of cellularinteraction with a pure crosslinked hyaluronan material throughintroduction of a silk fibroin hydrogel component. This increasedcellularity at the implant site could ultimately correlate to analternative host response to the hyaluronan including awound-healing-type response involving neo-collagen deposition duringimplant biorseorption. Taken together, the data here suggest that thecombination hyaluronan/silk fibroin hydrogel material not only acts as adermal filler for intradermal defects, but also encourages neo-collagendeposition through a native healing response.

Example 22 Use of Dermal Filler Composition for Treating a Facial Defectof the Cheek

This example illustrates the use of compositions and methods disclosedherein for treating a facial defect of the cheek.

A 28-year-old woman presents with a lean face. She felt her face lookedold, sad and bitter because of the less fullness of her check contour.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for softtissue treatment using the compositions and methods disclosed herein. Acomposition comprising silk fibroin hydrogel component and a hyaluronancomponent is administered subcutaneously and under superficialmusculoaponeurotix system into the checks regions; about 15 mL ofcomposition into the left and right cheeks. The individual is monitoredfor approximately 7 days. The physician evaluates the cheeks tissue anddetermines that the treatment was successful. Both the woman and herphysician are satisfied with the results of the procedure because shelooked younger. Approximately one month after the procedure, the womanindicates that his quality of life has improved.

Example 23 Use of Dermal Filler Composition for Treating FacialImperfection of Eyelids

This example illustrates the use of compositions and methods disclosedherein for treating a facial imperfection of the eyelids.

A 37-year-old woman presents with sunken eyes and this appearance madeher look old and fierce. Pre-operative evaluation of the person includesroutine history and physical examination in addition to thoroughinformed consent disclosing all relevant risks and benefits of theprocedure. The physician evaluating the individual determines that sheis a candidate for soft tissue treatment using the compositions andmethods disclosed herein. A composition comprising silk fibroin hydrogelcomponent and a hyaluronan component is administered subcutaneously andunder superficial musculoaponeurotix system into the upper eyelidregions; about 2.5 mL of composition into the left and right eyelidregions. The individual is monitored for approximately 7 days. Thephysician evaluates the eyelid regions and determines that the treatmentwas successful. Both the woman and her physician are satisfied with theresults of the procedure because she looked younger. Approximately onemonth after the procedure, the woman indicates that his quality of lifehas improved.

Example 24 Use of Dermal Filler Composition for Treating Wrinkles

This example illustrates the use of compositions and methods disclosedherein for treating wrinkles.

A 55-year-old woman presents with wrinkles around the eyes and cheekareas. Pre-operative evaluation of the person includes routine historyand physical examination in addition to thorough informed consentdisclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor soft tissue treatment using the compositions and methods disclosedherein. A composition comprising silk fibroin hydrogel component and ahyaluronan component is administered subcutaneously and undersuperficial musculoaponeurotix system into the upper eyelid and cheekregions; about 1.5 mL of composition into the left and right eyelid andcheek regions. The individual is monitored for approximately 7 days. Thephysician evaluates the facial regions and determines that the treatmentwas successful. Both the woman and her physician are satisfied with theresults of the procedure because she looked younger. Approximately onemonth after the procedure, the woman indicates that his quality of lifehas improved.

Example 25 Use of Dermal Filler Composition for Treating a Breast Defect

This example illustrates the use of compositions and methods disclosedherein for treating a breast defect.

A 32-year-old woman presents with complaints that the medial portions ofher breast implants are visible, which accentuated the “bony” appearanceof her sternum. In addition she felt her breast are too far apart.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for softtissue treatment using the compositions and methods disclosed herein. Acomposition comprising silk fibroin hydrogel component and a hyaluronancomponent is administered subcutaneously over the lateral sternum andmedial breast bilaterally, 15 mL on the right and 10 mL on the left. Thecomposition is administered in a tear like fashion to increase thesurface area to volume ratio. The individual is monitored forapproximately 7 days. The physician evaluates the breasts and determinesthat the treatment was successful. Both the woman and her physician aresatisfied with the results of the procedure. Approximately one monthafter the procedure, the woman indicates that his quality of life hasimproved.

Example 26 Use of Dermal Filler Composition for Breast Augmentation

This example illustrates the use of compositions and methods disclosedherein for breast augmentation.

A 28-year-old woman presents micromastia or breast hypoplasia.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for softtissue treatment using the compositions and methods disclosed herein. Acomposition comprising silk fibroin hydrogel component and a hyaluronancomponent is administered subcutaneously using axillary, periareolar,and inframammary routes bilaterally, 90 mL on the right and 145 mL onthe left. The composition is administered in a tear like fashion toincrease the surface area to volume ratio. The individual is monitoredfor approximately 7 days. The physician evaluates the breasts anddetermines that the treatment was successful. Both the woman and herphysician are satisfied with the results of the procedure. Approximatelyone month after the procedure, the woman indicates that his quality oflife has improved.

Example 27 Adipose Tissue Transplant for Breast Disorder

This example illustrates the use of compositions and methods disclosedherein for treating a breast disorder.

A 29-year-old woman presents with bilaterial tiberous breast deformity.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for softtissue treatment using the compositions and methods disclosed herein. Acomposition comprising silk fibroin hydrogel component and a hyaluronancomponent is administered subcutaneously in multiple planes axillary,periareolar, and inframammary routes bilaterally, 180 mL on the rightand 170 mL on the left. The composition is administered in a tear likefashion to increase the surface area to volume ratio. The individual ismonitored for approximately 7 days. The physician evaluates the breastsand determines that the treatment was successful. Both the woman and herphysician are satisfied with the results of the procedure. Approximatelyone month after the procedure, the woman indicates that his quality oflife has improved.

In closing, it is to be understood that although aspects of the presentspecification have been described with reference to the variousembodiments, one skilled in the art will readily appreciate that thespecific examples disclosed are only illustrative of the principles ofthe subject matter disclosed herein. Therefore, it should be understoodthat the disclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used herein,the term “about” means that the item, parameter or term so qualifiedencompasses a range of plus or minus ten percent above and below thevalue of the stated item, parameter or term. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thespecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

1. A composition comprising a gel phase, the gel phase including a)hydrogel particles comprising a substantially sericin-depleted silkfibroin and an amphiphilic peptide; and b) hydrogel particles comprisinga matrix polymerand,
 2. The composition of claim 1, wherein the silkfibroin hydrogel particles comprises about 1% (w/v) to about 10% (w/v)of silk fibroin.
 3. The composition of claim 1, wherein the finalconcentration of the silk fibroin is from about 3 mg/g to about 30 mg/g.4. The composition of claim 1, wherein the amphiphilic peptidecomprising a RGD motif or a non-RDG integrin.
 5. The composition ofclaim 3, wherein the amphiphilic peptide is 23 RGD.
 6. The compositionof claim 1, wherein the amphiphilic peptide comprises of a tail region,followed by a spacer region and finally a RGD motif.
 7. The compositionof claim 1, wherein the silk fibroin hydrogel particles comprises amolar ratio of 1:10 to 10:1 moles of the amphiphilic peptide per mole ofthe silk fibroin.
 8. The composition of claim 6, wherein the silkfibroin hydrogel particles comprises a molar ratio of 3:1 moles of theamphiphilic peptide per mole of the silk fibroin.
 9. The composition ofclaim 1, wherein the silk fibroin hydrogel particles comprises a proteinstructure having a β-sheet conformation of at least 80%.
 10. Thecomposition of claim 1, wherein the silk fibroin hydrogel particlescomprises a protein structure having a β-sheet conformation of at least50%.
 11. The composition of claim 1, wherein the silk fibroin hydrogelparticles comprises a protein structure having a β-sheet conformation ofat least 20%.
 12. The composition of claim 1, wherein the silk fibroinhydrogel particles comprises a protein structure having an α-helical andrandom coil conformation of at most 20%.
 13. The composition of claim 1,wherein the silk fibroin hydrogel particles further comprise a syntheticmolecule having the formula: (molecule X)_(n)—(spacerpeptide)₀₋₃₀₀−(five-amino-acid peptide tail) is conjugated to the silkfibroin.
 14. The composition of claim 1, wherein the matrix polymer is aglycosaminoglycan, a lubricin, a polysaccharide, or any combinationthereof.
 15. The composition of claim 14, wherein the glycosaminoglycanis a chondroitin sulfate, a dermatan sulfate, a keratan sulfate, aheperan sulfate, a hyaluronan, or any combination thereof.
 16. Thecomposition of claim 14, wherein the polysaccharide is a dextran, adextrin, a starch, a hetastarch, a glycogen, a polyvinyl acetate, apolyvinyl pyrrolidone, a polyethylene glycol, a polyethylene imine, acellulose, a methyl cellulose, a carboxymethyl cellulose, a hydroxyethylcellulose, a hydroxypropyl cellulose, a hydroxyethyl methyl cellulose, ahydroxypropyl methyl cellulose, or any combination thereof.
 17. Thecomposition of claim 1, wherein the matrix polymer is crosslinked. 18.The composition of claim 17, wherein the crosslinked matrix polymer hasa degree of crosslinking of at least 1%.
 19. The composition of claim17, wherein the crosslinked matrix polymer has a degree of crosslinkingof at most 17%.
 20. The composition of claim 17, wherein the crosslinkedmatrix polymer has a degree of crosslinking of about 1% to about 17%.21. The composition of claim 1, wherein the uncrosslinked matrix polymerrepresents about 90% or more by weight of the total matrix polymerpresent in the composition.
 22. The composition of claim 1, wherein thefinal concentration of the matrix polymer is from about 12 mg/g to about22.8 mg/g.
 23. The composition of claim 1, wherein the silk fibroinhydrogel particles and matrix polymer hydrogel particles have across-sectional area from about 0.1 μm² to about 1000 μm².
 24. Thecomposition of claim 1, wherein the silk fibroin hydrogel particles andmatrix polymer hydrogel particles have a cross-sectional area from about0.1 μm² to about 10 μm².
 25. The composition of claim 1, wherein thesilk fibroin hydrogel particles and matrix polymer hydrogel particleshave a cross-sectional area from about 20 μm² to about 50 μm².
 26. Thecomposition of claim 1, wherein the composition further comprises acarrier phase.
 27. The composition of claim 26, wherein the carrierphase comprises saline.
 28. The composition of claim 26, wherein thecarrier phase comprises a surfactant solution.
 29. The composition ofclaim 26, wherein the gel phase is 50% to 99% of the total formulationvolume, the remainder being a carrier solution.
 30. The composition ofclaim 29, wherein the gel phase is 75% of the total formulation volume,the remainder being a carrier solution.
 31. The composition of claim 1,wherein the composition further comprising lidocaine.
 32. Thecomposition of claim 1, wherein, upon injection, the hydrogel particlesremains substantially at the injection site for one month to eighteenmonths.
 33. A composition comprising a gel phase, the gel phaseincluding hydrogel particles comprising a substantially sericin-depletedsilk fibroin and a matrix polymer.
 34. The composition of claim 33,wherein the matrix polymer is a glycosaminoglycan, a lubricin, apolysaccharide, or any combination thereof.
 35. The composition of claim34, wherein the glycosaminoglycan is a chondroitin sulfate, a dermatansulfate, a keratan sulfate, a heperan sulfate, a hyaluronan, or anycombination thereof.
 36. The composition of claim 34, wherein thepolysaccharide is a dextran, a dextrin, a starch, a hetastarch, aglycogen, a polyvinyl acetate, a polyvinyl pyrrolidone, a polyethyleneglycol, a polyethylene imine, a cellulose, a methyl cellulose, acarboxymethyl cellulose, a hydroxyethyl cellulose, a hydroxypropylcellulose, a hydroxyethyl methyl cellulose, a hydroxypropyl methylcellulose, or any combination thereof.
 37. The composition of claim 33,wherein the matrix polymer is crosslinked.
 38. The composition of claim33, wherein the crosslinked matrix polymer has a degree of crosslinkingof at least 1%.
 39. The composition of claim 33, wherein the crosslinkedmatrix polymer has a degree of crosslinking of at most 17%.
 40. Thecomposition of claim 33, wherein the crosslinked matrix polymer has adegree of crosslinking of about 1% to about 17%.
 41. The composition ofclaim 33, wherein the uncrosslinked matrix polymer represents about 90%or more by weight of the total matrix polymer present in thecomposition.
 42. The composition of claim 33, wherein the finalconcentration of the matrix polymer is from about 12 mg/g to about 22.8mg/g.
 43. The composition of claim 33, wherein the hydrogel particlescomprises about 1% (w/v) to about 10% (w/v) of silk fibroin.
 44. Thecomposition of claim 33, wherein the final concentration of the silkfibroin is from about 3 mg/g to about 30 mg/g.
 45. The composition ofclaim 33, wherein the hydrogel particles further comprise an amphiphilicpeptide.
 46. The composition of claim 33, wherein the silk fibroinhydrogel particles and matrix polymer hydrogel particles have across-sectional area from about 0.1 μm² to about 1000 μm².
 47. Thecomposition of claim 33, wherein the silk fibroin hydrogel particles andmatrix polymer hydrogel particles have a cross-sectional area from about0.1 μm² to about 10 μm².
 48. The composition of claim 33, wherein thesilk fibroin hydrogel particles and matrix polymer hydrogel particleshave a cross-sectional area from about 20 μm² to about 50 μm².
 49. Thecomposition of claim 33, wherein the composition further comprises acarrier phase.
 50. The composition of claim 49, wherein the carrierphase comprises saline.
 51. The composition of claim 49, wherein thecarrier phase comprises a surfactant solution.
 52. The composition ofclaim 49, wherein the gel phase is 50% to 99% of the total formulationvolume, the remainder being a carrier solution.
 53. The composition ofclaim 52, wherein the gel phase is 75% of the total formulation volume,the remainder being a carrier solution.
 54. The composition of claim 33,wherein the composition further comprising lidocaine.
 55. Thecomposition of claim 33, wherein, upon injection, the hydrogel particlesremains substantially at the injection site for one month to eighteenmonths.
 56. A method of treating a soft tissue condition in anindividual in need thereof, the method comprising the step ofadministering a composition of claim 1 into a skin region of theindividual, wherein the administration improves the condition.
 57. Themethod of claim 56, wherein the soft tissue condition is a breast tissuecondition, a facial tissue condition, a neck condition, a skincondition, an upper arm condition, a lower arm condition, a handcondition, a shoulder condition, a back condition, a torso includingabdominal condition, a buttock condition, an upper leg condition, alower leg condition including calf condition, a foot condition includingplantar fat pad condition, an eye condition, a genital condition, or acondition effecting another body part, region or area.
 58. The method ofclaim 57, wherein the breast tissue condition is a breast imperfection,a breast defect, a breast augmentation, or a breast reconstruction. 59.The method of claim 57, wherein the facial tissue condition is a facialimperfection, a facial defect, a facial augmentation, or a facialreconstruction.
 60. The method of claim 57, wherein the facial tissuecondition is a dermal divot, a sunken check, a thin lip, a nasalimperfection or defect, a retro-orbital imperfection or defect, a facialfold, a facial line, a facial wrinkle, or other size, shape or contourimperfection or defect of the face.
 61. The method of claim 60, whereinthe wrinkle is a glabellar line, a nasolabial line, a perioral line, ora marionette line.
 62. The method of claim 57, wherein the facial tissuecondition is skin dehydration, a lack of skin elasticity, skinroughness, a lack of skin tautness, a skin stretch line or mark, or skinpaleness.
 63. The method of claim 56, wherein the soft tissue conditionis Parry-Romberg syndrome or lupus erythematosus profundus.
 64. Themethod of claim 56, wherein the soft tissue condition is urinaryincontinence, fecal incontinence, or gastroesophageal reflux disease(GERD).
 65. A method of treating a soft tissue condition in anindividual in need thereof, the method comprising the step ofadministering a composition of claim 33 into a skin region of theindividual, wherein the administration improves the condition.
 66. Themethod of claim 65, wherein the soft tissue condition is a breast tissuecondition, a facial tissue condition, a neck condition, a skincondition, an upper arm condition, a lower arm condition, a handcondition, a shoulder condition, a back condition, a torso includingabdominal condition, a buttock condition, an upper leg condition, alower leg condition including calf condition, a foot condition includingplantar fat pad condition, an eye condition, a genital condition, or acondition effecting another body part, region or area.
 67. The method ofclaim 66, wherein the breast tissue condition is a breast imperfection,a breast defect, a breast augmentation, or a breast reconstruction. 68.The method of claim 66, wherein the facial tissue condition is a facialimperfection, a facial defect, a facial augmentation, or a facialreconstruction.
 69. The method of claim 66, wherein the facial tissuecondition is a dermal divot, a sunken check, a thin lip, a nasalimperfection or defect, a retro-orbital imperfection or defect, a facialfold, a facial line, a facial wrinkle, or other size, shape or contourimperfection or defect of the face.
 70. The method of claim 69, whereinthe wrinkle is a glabellar line, a nasolabial line, a perioral line, ora marionette line.
 71. The method of claim 66, wherein the facial tissuecondition is skin dehydration, a lack of skin elasticity, skinroughness, a lack of skin tautness, a skin stretch line or mark, or skinpaleness.
 72. The method of claim 65, wherein the soft tissue conditionis Parry-Romberg syndrome or lupus erythematosus profundus.
 73. Themethod of claim 65, wherein the soft tissue condition is urinaryincontinence, fecal incontinence, or gastroesophageal reflux disease(GERD).